Modulation of gene expression and screening for deregulated protein expression

ABSTRACT

Disclosed herein include compositions and methods of modulating protein expression that utilizes an activator or a repressor of a non-sense mediated RNA decay switch exon (NSE). In some embodiments, also included herein are compositions and methods of modulating protein expression that uses an agent that targets a transposed element.

CROSS-REFERENCE

This application claims the benefit of UK Patent Application No:1517937.7, filed on Oct. 9, 2015, and UK Patent Application No:1614744.9, filed on Aug. 31, 2016, each of which are incorporated hereinby reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 1, 2016, isnamed 47991-718 601 SL.txt and is 34,863 bytes in size.

BACKGROUND OF THE INVENTION

The ATM protein belongs to the PI3/PI4-kinase family and is involved inthe developments of the nervous system and the immune system. The ATMprotein kinase is activated upon DNA damage and subsequently coordinatesthe DNA repair mechanism.

SUMMARY OF THE INVENTION

In certain embodiments, described herein include methods of screening asubject susceptible to functional-ATM protein deficiency and associatedconditions, methods for selecting subjects for treatment, methods fortreatment or prevention of conditions associated with functional-ATMprotein deficiency, methods of modifying a cells susceptibility to DNAdamaging radio- and chemotherapy, methods for treatment of cancer, andassociated compositions and kits.

Disclosed herein, in certain embodiments, is a method of screening asubject for susceptibility to functional-ATM protein deficiency, whereinthe screening comprises determining the presence of a non-thyminevariant residue rs609261 located at position −3 relative to the 3′splice site of NSE (cryptic exon in ATM intron 28) of the human genome,wherein the presence of a non-thymine variant residue rs609261 indicatesthat the subject has, or is susceptible to, functional-ATM proteindeficiency. In some embodiments, the NSE comprises a sequence comprisingtctacaggttggctgcatagaagaaaaag (SEQ ID NO: 57). In some embodiments, theNSE repressor agent binds to the NSE within a sequence comprisingagTCTACAGGTTGGCTGCATAGAAGAAAAAGgtagag (SEQ ID NO: 58); ortcttagTCTACAGGTTGGCTGCATAGAAGAAAAAGgtagag (SEQ ID NO: 59); ortctcagTCTACAGGTTGGCTGCATAGAAGAAAAAGgtagag (SEQ ID NO: 60). In someembodiments, the NSE repressor agent binds to the NSE or its 5′ or 3′splice site in ATM intron 28 of the NSE. In some embodiments, the NSErepressor agent and/or NSE activator agent comprises a polynucleic acidpolymer. In some embodiments, the NSE repressor agent and/or NSEactivator agent is an SSO (Splice Switching Oligonucleotide). In someembodiments, the NSE repressor agent and/or NSE activator agent isassociated with a delivery vehicle suitable for delivering the NSErepressor agent and/or NSE activator agent to cells. In someembodiments, the NSE repressor agent comprises: an SSO of the sequencecuucuaugcagccaaccuguagacu (SEQ ID NO: 53) (SSO—NSE3), or a nucleic acidanalogue thereof; or an SSO of the sequence accuuuuucuucuaugcagccaac(SEQ ID NO: 54) (SSO—NSE5), or a nucleic acid analogue thereof; and/orthe NSE repressor agent comprises or consists of any one SSO selectedfrom the group comprising: aacauuucuauuuaguuaaaagc (SEQ ID NO: 23) (SSOA11); uuaguauuccuugacuuua (SEQ ID NO: 26) (SSO A17);gacugguaaauaauaaacauaauuc (SEQ ID NO: 37) (SSO B2);auauauuagagauacaucagcc (SEQ ID NO: 39) (SSO B4); anduuagagaaucauuuuaaauaagac (SEQ ID NO: 51) (SSO AN3), or combinationsthereof.

Disclosed herein, in certain embodiments, is a method of selecting asubject for treatment, wherein the subject is susceptible to afunctional-ATM protein deficiency, the method comprising determining thepresence of a non-thymine variant residue rs609261 located at position−3 relative to the 3′ splice site of NSE (cryptic exon in ATM intron 28)of the human genome, wherein the presence of a non-thymine variantresidue rs609261 indicates that the subject has, or is susceptible to, afunctional-ATM protein deficiency, and selecting such subject fortreatment with an agent thereby increasing a functional-ATM levels inthe subject. In some embodiments, the method further comprisesadministering the agent for treatment of the selected subject. In someembodiments, the agent comprises a NSE repressor agent. In someembodiments, the NSE comprises a sequence comprisingtctacaggttggctgcatagaagaaaaag (SEQ ID NO: 57). In some embodiments, theNSE repressor agent binds to the NSE within a sequence comprisingagTCTACAGGTTGGCTGCATAGAAGAAAAAGgtagag (SEQ ID NO: 58); ortcttagTCTACAGGTTGGCTGCATAGAAGAAAAAGgtagag (SEQ ID NO: 59); ortctcagTCTACAGGTTGGCTGCATAGAAGAAAAAGgtagag (SEQ ID NO: 60). In someembodiments, the NSE repressor agent binds to the NSE or its 5′ or 3′splice site in ATM intron 28 of the NSE. In some embodiments, the NSErepressor agent and/or NSE activator agent comprises a polynucleic acidpolymer. In some embodiments, the NSE repressor agent and/or NSEactivator agent is an SSO (Splice Switching Oligonucleotide). In someembodiments, the NSE repressor agent and/or NSE activator agent isassociated with a delivery vehicle suitable for delivering the NSErepressor agent and/or NSE activator agent to cells. In someembodiments, the NSE repressor agent comprises: an SSO of the sequencecuucuaugcagccaaccuguagacu (SEQ ID NO: 53) (SSO—NSE3), or a nucleic acidanalogue thereof; or an SSO of the sequence accuuuuucuucuaugcagccaac(SEQ ID NO: 54) (SSO—NSE5), or a nucleic acid analogue thereof; and/orthe NSE repressor agent comprises or consists of any one SSO selectedfrom the group comprising: aacauuucuauuuaguuaaaagc (SEQ ID NO: 23) (SSOA11); uuaguauuccuugacuuua (SEQ ID NO: 26) (SSO A17);gacugguaaauaauaaacauaauuc (SEQ ID NO: 37) (SSO B2);auauauuagagauacaucagcc (SEQ ID NO: 39) (SSO B4); anduuagagaaucauuuuaaauaagac (SEQ ID NO: 51) (SSO AN3), or combinationsthereof.

Disclosed herein, in certain embodiments, is a method of treatment orprevention of a functional-ATM protein deficiency in a subject, themethod comprising identifying a presence of a non-thymine variantresidue rs609261 located at position -3 relative to the 3′ splice siteof NSE (cryptic exon in ATM intron 28) of the human genome, wherein thepresence of a non-thymine variant residue rs609261 indicates that thesubject has, or is susceptible to, a functional-ATM protein deficiency,and administration of an agent to the subject, which is arranged toincrease functional-ATM levels. In some embodiments, the NSE comprises asequence comprising tctacaggttggctgcatagaagaaaaag (SEQ ID NO: 57). Insome embodiments, the NSE repressor agent binds to the NSE within asequence comprising agTCTACAGGTTGGCTGCATAGAAGAAAAAGgtagag (SEQ ID NO:58); or tcttagTCTACAGGTTGGCTGCATAGAAGAAAAAGgtagag (SEQ ID NO: 59); ortctcagTCTACAGGTTGGCTGCATAGAAGAAAAAGgtagag (SEQ ID NO: 60). In someembodiments, the NSE repressor agent binds to the NSE or its 5′ or 3′splice site in ATM intron 28 of the NSE. In some embodiments, the NSErepressor agent and/or NSE activator agent comprises a polynucleic acidpolymer. In some embodiments, the NSE repressor agent and/or NSEactivator agent is an SSO (Splice Switching Oligonucleotide). In someembodiments, the NSE repressor agent and/or NSE activator agent isassociated with a delivery vehicle suitable for delivering the NSErepressor agent and/or NSE activator agent to cells. In someembodiments, the NSE repressor agent comprises: an SSO of the sequencecuucuaugcagccaaccuguagacu (SEQ ID NO: 53) (SSO—NSE3), or a nucleic acidanalogue thereof; or an SSO of the sequence accuuuuucuucuaugcagccaac(SEQ ID NO: 54) (SSO—NSE5), or a nucleic acid analogue thereof; and/orthe NSE repressor agent comprises or consists of any one SSO selectedfrom the group comprising: aacauuucuauuuaguuaaaagc (SEQ ID NO: 23) (SSOA11); uuaguauuccuugacuuua (SEQ ID NO: 26) (SSO A17);gacugguaaauaauaaacauaauuc (SEQ ID NO: 37) (SSO B2);auauauuagagauacaucagcc (SEQ ID NO: 39) (SSO B4); anduuagagaaucauuuuaaauaagac (SEQ ID NO: 51) (SSO AN3), or combinationsthereof.

Disclosed herein, in certain embodiments, is a method of treatment orprevention of a condition associated with a functional-ATM proteindeficiency, comprising the administration of a NSE repressor agentthereby increasing a functional ATM protein level, wherein the agentbinds to a NSE in ATM intron 28 of a pre-mRNA transcript therebydecreasing inclusion of the NSE in the mature RNA transcript. In someembodiments, the decreasing inclusion of the NSE in the mature RNAtranscript provides an increase in functional ATM protein expression. Insome embodiments, the method is for treatment or prevention offunctional-ATM protein deficiency in a subject or an at-risk populationof subjects is for treatment or prevention of a condition or symptomsassociated with a functional-ATM protein deficiency. In someembodiments, the condition is ataxia-telangiectasia; cancer; immunedeficiency; cellular radiosensitivity; or chromosomal instability. Insome embodiments, the NSE comprises a sequence comprisingtctacaggttggctgcatagaagaaaaag (SEQ ID NO: 57). In some embodiments, theNSE repressor agent binds to the NSE within a sequence comprisingagTCTACAGGTTGGCTGCATAGAAGAAAAAGgtagag (SEQ ID NO: 58); ortcttagTCTACAGGTTGGCTGCATAGAAGAAAAAGgtagag (SEQ ID NO: 59); ortctcagTCTACAGGTTGGCTGCATAGAAGAAAAAGgtagag (SEQ ID NO: 60). In someembodiments, the NSE repressor agent binds to the NSE or its 5′ or 3′splice site in ATM intron 28 of the NSE. In some embodiments, the NSErepressor agent and/or NSE activator agent comprises a polynucleic acidpolymer. In some embodiments, the NSE repressor agent and/or NSEactivator agent is an SSO (Splice Switching Oligonucleotide). In someembodiments, the NSE repressor agent and/or NSE activator agent isassociated with a delivery vehicle suitable for delivering the NSErepressor agent and/or NSE activator agent to cells. In someembodiments, the NSE repressor agent comprises: an SSO of the sequencecuucuaugcagccaaccuguagacu (SEQ ID NO: 53) (SSO—NSE3), or a nucleic acidanalogue thereof; or an SSO of the sequence accuuuuucuucuaugcagccaac(SEQ ID NO: 54) (SSO—NSE5), or a nucleic acid analogue thereof; and/orthe NSE repressor agent comprises or consists of any one SSO selectedfrom the group comprising: aacauuucuauuuaguuaaaagc (SEQ ID NO: 23) (SSOA11); uuaguauuccuugacuuua (SEQ ID NO: 26) (SSO A17);gacugguaaauaauaaacauaauuc (SEQ ID NO: 37) (SSO B2);auauauuagagauacaucagcc (SEQ ID NO: 39) (SSO B4); anduuagagaaucauuuuaaauaagac (SEQ ID NO: 51) (SSO AN3), or combinationsthereof.

Disclosed herein, in certain embodiments, is a method of treatment orprevention of a condition associated with deregulation of ATM expressionin a subject comprising administering a NSE-activator agent to thesubject, wherein the NSE-activator agent increases inclusion of a NSE inan ATM mature RNA transcript by binding to a regulatory motif in ATMintron 28, or by binding to a U2AF65 binding site upstream of apseudoexon located 3′ of a NSE in ATM intron 28 of an ATM pre-mRNAtranscript. In some embodiments, disclosed herein is a method oftreatment or prevention of a condition associated with deregulation ofATM expression in a subject comprising administering a NSE-activatoragent to the subject, wherein the NSE-activator agent increasesinclusion of a NSE in an ATM mature RNA transcript by binding to aregulatory motif in ATM intron 28, optionally wherein the regulatorymotifs in ATM intron 28 compete with NSE for spliceosomal components,and further optionally wherein such motifs comprise a 24 nucleotidepseudoexon (PE) located 3′ of NSE in ATM intron 28 of the pre-mRNAtranscript or binding to a U2AF65 binding site upstream of thepseudoexon. In some embodiments, increasing inclusion of the NSE in themature RNA transcript provides a decrease in functional ATM proteinexpression. In some embodiments, the pseudoexon comprises the sequencetcatcgaatacttttggaaataag (SEQ ID NO: 61). In some embodiments, theregulatory motif in ATM intron 28 competes with the NSE for spliceosomalcomponents. In some embodiments, the regulatory motif in ATM intron 28comprises a 24 nucleotide pseudoexon (PE) located 3′ of the NSE in ATMintron 28 of the pre-mRNA transcript. In some embodiments, the NSErepressor agent and/or NSE activator agent comprises a polynucleic acidpolymer. In some embodiments, the NSE repressor agent and/or NSEactivator agent is an SSO (Splice Switching Oligonucleotide). In someembodiments, the NSE repressor agent and/or NSE activator agent isassociated with a delivery vehicle suitable for delivering the NSErepressor agent and/or NSE activator agent to cells. In someembodiments, the NSE repressor agent comprises: an SSO of the sequencecuucuaugcagccaaccuguagacu (SEQ ID NO: 53) (SSO—NSE3), or a nucleic acidanalogue thereof; or an SSO of the sequence accuuuuucuucuaugcagccaac(SEQ ID NO: 54) (SSO—NSE5), or a nucleic acid analogue thereof; and/orthe NSE repressor agent comprises or consists of any one SSO selectedfrom the group comprising: aacauuucuauuuaguuaaaagc (SEQ ID NO: 23) (SSOA11); uuaguauuccuugacuuua (SEQ ID NO: 26) (SSO A17);gacugguaaauaauaaacauaauuc (SEQ ID NO: 37) (SSO B2);auauauuagagauacaucagcc (SEQ ID NO: 39) (SSO B4); anduuagagaaucauuuuaaauaagac (SEQ ID NO: 51) (SSO AN3), or combinationsthereof. In some embodiments, the NSE activator agent comprises the SSOPEkr/PEdel; and/or the NSE activator agent comprises or consists of anyone SSO selected from the group comprising: aacuuaaagguuauaucuc (SEQ IDNO: 18) (SSO A2); uauaaauacgaauaaaucga (SEQ ID NO: 19) (SSO A4);caacacgacauaaccaaa (SEQ ID NO: 21) (SSO A9); gguaugagaacuauagga (SEQ IDNO: 32) (SSO A23); gguaauaagugucacaaa (SEQ ID NO: 34) (SSOA25);guaucauacauuagaagg (SEQ ID NO: 35) (SSO A26); anduguggggugaccacagcuu (SEQ ID NO: 45) (SSO B11), or combinations thereof

Disclosed herein, in certain embodiments, is a method of treatment orprevention of cancer in a subject comprising administering aNSE-activator agent to the subject, wherein the NSE-activator agentincreases a cancer cell's susceptibility to cytotoxic therapy with DNAdamaging agents such as radiotherapy, wherein the NSE-activator agentincreases inclusion of a NSE in an ATM mature RNA transcript by bindingto a regulatory motif in ATM intron 28, or by binding to a U2AF65binding site upstream of a pseudoexon located 3′ of a NSE in ATM intron28 of an ATM pre-mRNA transcript, and treating the subject with thecytotoxic therapy, such as radiotherapy or chemotherapy. In someembodiments, disclosed herein is a method of treatment or prevention ofcancer in a subject comprising the administration of a NSE-activatoragent arranged to increase a cancer cell's susceptibility to cytotoxictherapy with DNA damaging agents such as radiotherapy, wherein theNSE-activator agent is arranged to increase NSE inclusion in ATM matureRNA transcript by binding to regulatory motifs in ATM intron 28,optionally wherein the regulatory motifs in ATM intron 28 compete withNSE for spliceosomal components, and further optionally wherein suchmotifs comprise a 24 nucleotide pseudoexon (PE) located 3′ of NSE in ATMintron 28 of the pre-mRNA transcript or binding to a U2AF65 binding siteupstream of the pseudoexon; and treating the subject with the cytotoxictherapy, such as radiotherapy or chemotherapy. In some embodiments,increasing inclusion of the NSE in the mature RNA transcript provides adecrease in functional ATM protein expression. In some embodiments, thepseudoexon comprises the sequence tcatcgaatacttttggaaataag (SEQ ID NO:61). In some embodiments, increasing inclusion of the NSE in the matureRNA transcript provides a decrease in functional ATM protein expression.In some embodiments, the pseudoexon comprises the sequencetcatcgaatacttttggaaataag (SEQ ID NO: 61). In some embodiments, theregulatory motif in ATM intron 28 competes with the NSE for spliceosomalcomponents. In some embodiments, the regulatory motif in ATM intron 28comprises a 24 nucleotide pseudoexon (PE) located 3′ of the NSE in ATMintron 28 of the pre-mRNA transcript. In some embodiments, the NSErepressor agent and/or NSE activator agent comprises a polynucleic acidpolymer. In some embodiments, the NSE repressor agent and/or NSEactivator agent is an SSO (Splice Switching Oligonucleotide). In someembodiments, the NSE repressor agent and/or NSE activator agent isassociated with a delivery vehicle suitable for delivering the NSErepressor agent and/or NSE activator agent to cells. In someembodiments, the NSE repressor agent comprises: an SSO of the sequencecuucuaugcagccaaccuguagacu (SEQ ID NO: 53) (SSO—NSE3), or a nucleic acidanalogue thereof; or an SSO of the sequence accuuuuucuucuaugcagccaac(SEQ ID NO: 54) (SSO—NSE5), or a nucleic acid analogue thereof; and/orthe NSE repressor agent comprises or consists of any one SSO selectedfrom the group comprising: aacauuucuauuuaguuaaaagc (SEQ ID NO: 23) (SSOA11); uuaguauuccuugacuuua (SEQ ID NO: 26) (SSO A17);gacugguaaauaauaaacauaauuc (SEQ ID NO: 37) (SSO B2);auauauuagagauacaucagcc (SEQ ID NO: 39) (SSO B4); anduuagagaaucauuuuaaauaagac (SEQ ID NO: 51) (SSO AN3), or combinationsthereof In some embodiments, the NSE activator agent comprises the SSOPEkr/PEdel; and/or the NSE activator agent comprises or consists of anyone SSO selected from the group comprising: aacuuaaagguuauaucuc (SEQ IDNO: 18) (SSO A2); uauaaauacgaauaaaucga (SEQ ID NO: 19) (SSO A4);caacacgacauaaccaaa (SEQ ID NO: 21) (SSO A9); gguaugagaacuauagga (SEQ IDNO: 32) (SSO A23); gguaauaagugucacaaa (SEQ ID NO: 34) (SSOA25);guaucauacauuagaagg (SEQ ID NO: 35) (SSO A26); anduguggggugaccacagcuu (SEQ ID NO: 45) (SSO B11), or combinations thereof

Disclosed herein, in certain embodiments, is a method of increasing acell's susceptibility to cytotoxic therapy with DNA damaging agents suchas radiotherapy or chemotherapy comprising reducing ATM proteinexpression by administering a NSE-activator agent, wherein theNSE-activator agent increases inclusion of a NSE in an ATM mature RNAtranscript by binding to motifs in ATM intron 28, or by binding to aU2AF65 binding site upstream of a pseudoexon located 3′ of a NSE in ATMintron 28 of an ATM pre-mRNA transcript. Disclosed herein, in certainembodiments, is a method of increasing a cell's susceptibility tocytotoxic therapy with DNA damaging agents such as radiotherapy orchemotherapy comprising reducing ATM protein expression byadministrating a NSE-activator agent arranged to increase NSE inclusionin ATM mature RNA transcript by binding to motifs in ATM intron 28,optionally wherein the regulatory motifs in ATM intron 28 compete withNSE for spliceosomal components, and further optionally wherein suchmotifs comprise a 24 nucleotide pseudoexon (PE) located 3′ of NSE in ATMintron 28 of the pre-mRNA transcript or binding to a U2AF65 binding siteupstream of the pseudoexon. In some embodiments, increasing inclusion ofthe NSE in the mature RNA transcript provides a decrease in functionalATM protein expression. In some embodiments, the pseudoexon comprisesthe sequence tcatcgaatacttttggaaataag (SEQ ID NO: 61). In someembodiments, the regulatory motif in ATM intron 28 competes with the NSEfor spliceosomal components. In some embodiments, the regulatory motifin ATM intron 28 comprises a 24 nucleotide pseudoexon (PE) located 3′ ofthe NSE in ATM intron 28 of the pre-mRNA transcript. In someembodiments, the NSE repressor agent and/or NSE activator agentcomprises a polynucleic acid polymer. In some embodiments, the NSErepressor agent and/or NSE activator agent is an SSO (Splice SwitchingOligonucleotide). In some embodiments, the NSE repressor agent and/orNSE activator agent is associated with a delivery vehicle suitable fordelivering the NSE repressor agent and/or NSE activator agent to cells.In some embodiments, the NSE repressor agent comprises: an SSO of thesequence cuucuaugcagccaaccuguagacu (SEQ ID NO: 53) (SSO—NSE3), or anucleic acid analogue thereof; or an SSO of the sequenceaccuuuuucuucuaugcagccaac (SEQ ID NO: 54) (SSO—NSE5), or a nucleic acidanalogue thereof; and/or the NSE repressor agent comprises or consistsof any one SSO selected from the group comprising:aacauuucuauuuaguuaaaagc (SEQ ID NO: 23) (SSO A11); uuaguauuccuugacuuua(SEQ ID NO: 26) (SSO A17); gacugguaaauaauaaacauaauuc (SEQ ID NO: 37)(SSO B2); auauauuagagauacaucagcc (SEQ ID NO: 39) (SSO B4); anduuagagaaucauuuuaaauaagac (SEQ ID NO: 51) (SSO AN3), or combinationsthereof. In some embodiments, the NSE activator agent comprises the SSOPEkr/PEdel; and/or the NSE activator agent comprises or consists of anyone SSO selected from the group comprising: aacuuaaagguuauaucuc (SEQ IDNO: 18) (SSO A2); uauaaauacgaauaaaucga (SEQ ID NO: 19) (SSO A4);caacacgacauaaccaaa (SEQ ID NO: 21) (SSO A9); gguaugagaacuauagga (SEQ IDNO: 32) (SSO A23); gguaauaagugucacaaa (SEQ ID NO: 34) (SSOA25);guaucauacauuagaagg (SEQ ID NO: 35) (SSO A26); anduguggggugaccacagcuu (SEQ ID NO: 45) (SSO B11), or combinations thereof.

Disclosed herein, in certain embodiments, is a method of tailoringfunctional ATM expression in a subject, cell or tissue, comprising theadministration of a NSE-activator agent and/or a NSE-repressor agentdescribed herein. In some embodiments, the NSE repressor agent and/orNSE activator agent comprise a polynucleic acid polymer. In someembodiments, the NSE repressor agent and/or NSE activator agent is anSSO (Splice Switching Oligonucleotide). In some embodiments, the NSErepressor agent and/or NSE activator agent is associated with a deliveryvehicle suitable for delivering the NSE repressor agent and/or NSEactivator agent to cells. In some embodiments, the NSE repressor agentcomprises: an SSO of the sequence cuucuaugcagccaaccuguagacu (SEQ ID NO:53) (SSO—NSE3), or a nucleic acid analogue thereof; or an SSO of thesequence accuuuuucuucuaugcagccaac (SEQ ID NO: 54) (SSO—NSE5), or anucleic acid analogue thereof; and/or the NSE repressor agent comprisesor consists of any one SSO selected from the group comprising:aacauuucuauuuaguuaaaagc (SEQ ID NO: 23) (SSO A11); uuaguauuccuugacuuua(SEQ ID NO: 26) (SSO A17); gacugguaaauaauaaacauaauuc (SEQ ID NO: 37)(SSO B2); auauauuagagauacaucagcc (SEQ ID NO: 39) (SSO B4); anduuagagaaucauuuuaaauaagac (SEQ ID NO: 51) (SSO AN3), or combinationsthereof; or the method of which the NSE activator agent comprises theSSO PEkr/PEdel; and/or the NSE activator agent comprises or consists ofany one SSO selected from the group comprising: aacuuaaagguuauaucuc (SEQID NO: 18) (SSO A2); uauaaauacgaauaaaucga (SEQ ID NO: 19) (SSO A4);caacacgacauaaccaaa (SEQ ID NO: 21) (SSO A9); gguaugagaacuauagga (SEQ IDNO: 32) (SSO A23); gguaauaagugucacaaa (SEQ ID NO: 34) (SSOA25);guaucauacauuagaagg (SEQ ID NO: 35) (SSO A26); anduguggggugaccacagcuu (SEQ ID NO: 45) (SSO B11), or combinations thereof

Disclosed herein, in certain embodiments, is use of rs609261 and/orrs4988000 genotyping to predict a subject's response to therapy forconditions associated with ATM deregulation.

Disclosed herein, in certain embodiments, is a composition comprisingthe NSE repressor agent and/or the NSE activator agent described herein.In some embodiments, the composition is a pharmaceutically acceptableformulation.

Disclosed herein, in certain embodiments, is a method of treatment orprevention of functional-ATM protein deficiency in a subject, the methodcomprising identifying the presence of a non-thymine variant residuers609261 located at position -3 relative to the 3′ splice site of NSE(cryptic exon in ATM intron 28) of the human genome, wherein thepresence of a non-thymine variant residue rs609261 indicates that thesubject has, or is susceptible to, functional-ATM protein deficiency,and administration of an agent to the subject, which is arranged toreplace the non-thymine variant residue rs609261 with a thymine residue.In some embodiments, replacing the non-thymine variant residue rs609261comprises administration of an agent to the subject, which is arrangedto replace the non-thymine variant residue rs609261 with a thymineresidue. In some embodiments, the agent for replacement of thenon-thymine residue is a genomic editing molecule. In some embodiments,the agent for replacement of the non-thymine residue is CRISPR-Cas9, ora functional equivalent thereof, together with an appropriate RNAmolecule arranged to target rs609261.

Disclosed herein, in certain embodiments, is a method of treatment orprevention of functional-ATM protein deficiency in a subject, the methodcomprising replacing a non-thymine variant residue rs609261 located atposition -3 relative to the 3′ splice site of NSE (cryptic exon in ATMintron 28) of the human genome with a thymine residue. In someembodiments, replacing the non-thymine variant residue rs609261comprises administration of an agent to the subject, which is arrangedto replace the non-thymine variant residue rs609261 with a thymineresidue. In some embodiments, the agent for replacement of thenon-thymine residue is a genomic editing molecule. In some embodiments,the agent for replacement of the non-thymine residue is CRISPR-Cas9, ora functional equivalent thereof, together with an appropriate RNAmolecule arranged to target rs609261.

Disclosed herein, in certain embodiments, is a method of treatment orprevention of functional-ATM protein deficiency in a subject, the methodcomprising identifying the presence of a guanine variant residue atrs4988000 of the human genome, wherein the presence of a guanine variantresidue at rs4988000 indicates that the subject has, or is susceptibleto, functional-ATM protein deficiency, and administration of an agent tothe subject, which is arranged to replace the guanine variant residue atrs4988000 with adenine. In some embodiments, replacing the guaninevariant residue at rs4988000 comprises administration of an agent to thesubject, which is arranged to replace the guanine variant residue atrs4988000 with an adenine residue. In some embodiments, the agent forreplacement of the guanine residue is a genomic editing molecule. Insome embodiments, the agent for replacement of the guanine residue isCRISPR-Cas9, or a functional equivalent thereof, together with anappropriate RNA molecule arranged to target rs4988000.

Disclosed herein, in certain embodiments, is a method of treatment orprevention of functional-ATM protein deficiency in a subject, the methodcomprising replacing a guanine variant residue at rs4988000 of the humangenome with an adenine residue; or blocking the guanine residue by thebinding of an SSO. In some embodiments, replacing the guanine variantresidue at rs4988000 comprises administration of an agent to thesubject, which is arranged to replace the guanine variant residue atrs4988000 with an adenine residue. In some embodiments, the agent forreplacement of the guanine residue is a genomic editing molecule. Insome embodiments, the agent for replacement of the guanine residue isCRISPR-Cas9, or a functional equivalent thereof, together with anappropriate RNA molecule arranged to target rs4988000.

Disclosed herein, in certain embodiments, is a method of screening asubject or a population of subjects for susceptibility to functional-ATMprotein deficiency, wherein the screening comprises determining thepresence of a guanine variant residue at rs4988000 of the human genome,wherein the presence of a guanine variant residue at rs4988000 indicatesthat the subject (or group of subjects) has, or is susceptible to,functional-ATM protein deficiency.

Disclosed herein, in certain embodiments, is a method of selecting asubject or a population of subjects for treatment or prophylaxis,wherein the subject is susceptible to functional-ATM protein deficiency,the method comprising determining a presence of a guanine variantresidue at rs4988000 of a human subject's genome, wherein the presenceof a guanine variant residue at rs4988000 indicates that the subjecthas, or is susceptible to, the functional-ATM protein deficiency, andselecting the subject for treatment with an agent that increasesfunctional-ATM levels in the subject.

Disclosed herein, in certain embodiments, is a method of treatment orprevention of functional-ATM protein deficiency in a subject, the methodcomprising identifying a presence of a guanine variant residue atrs4988000 of a human subject's genome, wherein the presence of a guaninevariant residue at rs4988000 indicates that the subject has, or issusceptible to, functional-ATM protein deficiency, and administering anagent to the subject, wherein the agent increases functional-ATM levels.

In some embodiments, one or more methods disclosed herein is incombination to modify a CG haplotype to TA.

In some embodiments, one or more methods disclosed herein is incombination to identify a CG haplotype in a subject, and optionallytreat or select the patient for treatment.

Disclosed herein, in certain embodiments, is a method of modifyingregulation of inclusion of a NSE in a mature RNA transcript, the methodcomprising inserting or deleting one or more splicing regulatory motifsupstream or downstream of the NSE that compete with the NSE forspliceosomal components, said one or more splicing regulatory motifscomprising a cryptic splice site or a pseudo-exon. In some embodiments,the insertion or the deletion of the one or more splicing regulatorymotifs is in genomic DNA of ATM intron 28. In some embodiments,insertion of the one or more splicing regulatory motifs causes areduction in the inclusion of the NSE in the mature RNA transcript. Insome embodiments, the deletion of the one or more splicing regulatorymotifs causes an increase in the inclusion of the NSE in the mature RNAtranscript. In some embodiments, the insertion or the deletion of theone or more splicing regulatory motifs comprises the use of genomeediting technology, such as CRISPR-Cas9.

Disclosed herein, in certain embodiments, is a method of modifyingregulation of expression of a functional protein, wherein the expressionof a functional protein is regulated by inclusion of a NSE in a matureRNA transcript of a gene encoding the functional protein, the methodcomprising inserting or deleting one or more splicing regulatory motifsupstream or downstream of the NSE that compete with the NSE forspliceosomal components, said one or more splicing regulatory motifscomprising cryptic splice sites or pseudo-exons. In some embodiments,the insertion or the deletion of the one or more splicing regulatorymotifs is in genomic DNA of ATM intron 28. In some embodiments,insertion of the one or more splicing regulatory motifs causes areduction in the inclusion of the NSE in the mature RNA transcript. Insome embodiments, the deletion of the one or more splicing regulatorymotifs causes an increase in the inclusion of the NSE in the mature RNAtranscript. In some embodiments, the insertion or the deletion of theone or more splicing regulatory motifs comprises the use of genomeediting technology, such as CRISPR-Cas9.

Disclosed herein, in certain embodiments, is a kit comprising one ormore oligonucleotide probes for identifying rs609261 and/or rs4988000variants. In some embodiments, the one or more oligonucleotide probesare primers for use in PCR amplifying a region of a nucleic acidcomprising the rs609261 and/or the rs4988000 variants.

Disclosed herein, in certain embodiments, is a vector comprising anucleic acid encoding a NSE activating agent and/or a NSE repressoragent.

Disclosed herein, in certain embodiments, is a method of screening foran agent capable of modifying regulation of a gene's expressioncomprising: identifying a nonsense-mediated RNA decay switch exon (NSE)that limits functional gene expression; identifying one or more splicingregulatory motifs upstream or downstream of the NSE that compete withthe NSE for spliceosomal components, said regulatory motifs comprisingcryptic splice sites or pseudoexons; targeting the one or more splicingregulatory motifs with an antisense polynucleic acid comprising asequence that hybridizes to a splicing regulatory motif of the one ormore splicing regulatory motifs through Watson-Crick base pairing; anddetermining if there is an increased or decreased inclusion of the NSEin a mature RNA transcript of the gene.

Disclosed herein, in certain embodiments, is a method of modulatingexpression of a gene comprising providing an agent that binds to asplicing regulatory motif, such as a cryptic splice site or apseudoexon, that competes with a nonsense-mediated RNA decay switch exon(NSE) for spliceosomal components.

Disclosed herein, in certain embodiments, is an agent that binds to agene splicing regulatory motif, such as a cryptic splice site or apseudoexon, that competes with a nonsense-mediated RNA decay switch exon(NSE) for spliceosomal components, wherein the gene splicing regulatorymotif controls inclusion of the NSE into a mature RNA transcript of thegene.

Disclosed herein, in certain embodiments, is a method of modulatingprotein expression comprising: (a) contacting an isolated polynucleicacid polymer to a target cell of a subject; (b) hybridizing thecontacted polynucleic acid polymer to a target motif on a pre-processedmRNA transcript, wherein a hybridization of the contacted polynucleicacid polymer to the target motif either promotes or represses activationof a non-sense mediated RNA decay switch exon (NSE); (c) processing amRNA transcript of the pre-processed mRNA transcript, wherein the NSE iseither present or absent in the mRNA transcript; and (d) translating theprocessed mRNA transcript of step c), wherein the presence or absence ofthe NSE modulates protein expression. In some embodiments, the proteinis expressed from the processed mRNA transcript. In some embodiments,the presence of the NSE downregulates protein expression. In someembodiments, the absence of the NSE upregulates protein expression. Insome embodiments, the polynucleic acid polymer hybridizes to a motifwithin ATM intron 28. In some embodiments, the motif is a splicingregulatory motif that competes with the NSE for a spliceosomalcomponent. In some embodiments, the splicing regulatory motif comprisesa cryptic splice site or a pseudoexon. In some embodiments, thepseudoexon is a 24 nucleotide pseudoexon located at 3′ of a NSE in ATMintron 28 of the pre-mRNA transcript. In some embodiments, the motif isa U2AF65 binding site. In some embodiments, the motif is a motif withina transposed element, upstream of a transposed element, or downstream ofa transposed element. In some embodiments, the transposed element is Aluor MER51. In some embodiments, the isolated polynucleic acid polymerhybridizes to a target motif within Alu. In some embodiments, theisolated polynucleic acid polymer hybridizes to a target motif that iseither upstream or downstream of Alu. In some embodiments, the isolatedpolynucleic acid polymer hybridizes to a target motif downstream ofMER51. In some embodiments, the polynucleic acid polymer is from about10 to about 50 nucleotides in length. In some embodiments, the isolatedpolynucleic acid polymer comprises a sequence with at least 70%, 75%,80%, 85%, 90%, 95%, or 99% sequence identity to a sequence selected fromthe group consisting of SEQ ID NOs: 18-52. In some embodiments, thepolynucleic acid polymer is modified at a nucleoside moiety, at aphosphate moiety, at a 5′ terminus, at a 3′ terminus, or a combinationthereof. In some embodiments, the polynucleic acid polymer comprises anartificial nucleotide. In some embodiments, the artificial nucleotide isselected from the group consisting of 2′-O-methyl, 2′-O-methoxyethyl(2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro,2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMA0E),2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), 2′-O-N-methylacetamido (2′-O-NMA), a locked nucleic acid(LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a1′,5′-anhydrohexitol nucleic acid (HNA), a morpholino, amethylphosphonate nucleotide, a thiolphosphonate nucleotide, and a2′-fluoro N3-P5′-phosphoramidite.

Disclosed herein, in certain embodiments, is a method of modulatingprotein expression comprising: (a) contacting an isolated polynucleicacid polymer to a target cell of a subject; (b) hybridizing thecontacted polynucleic acid polymer to a target motif within a transposedelement, wherein a hybridization of the contacted polynucleic acidpolymer to the target motif either promotes or represses activation of anon-sense mediated RNA decay switch exon (NSE); (c) processing a mRNAtranscript of the pre-processed mRNA transcript, wherein the NSE iseither present or absent in the mRNA transcript; and (d) translating theprocessed mRNA transcript of step c), wherein the presence or absence ofthe NSE modulates protein expression. In some embodiments, the proteinis expressed from the processed mRNA transcript. In some embodiments,the presence of the NSE downregulates protein expression. In someembodiments, the absence of the NSE upregulates protein expression. Insome embodiments, the transposed element is Alu or MER51. In someembodiments, the isolated polynucleic acid polymer comprises a sequencewith at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity toa sequence selected from the group consisting of SEQ ID NOs: 18-52. Insome embodiments, the polynucleic acid polymer is from about 10 to about50 nucleotides in length. In some embodiments, the isolated polynucleicacid polymer hybridizes to a target motif within Alu. In someembodiments, the isolated polynucleic acid polymer hybridizes to atarget motif that is either upstream or downstream of Alu. In someembodiments, the isolated polynucleic acid polymer hybridizes to atarget motif downstream of MER51. In some embodiments, activation of theNSE further induces exon skipping. In some embodiments, the NSE islocated in intron 28. In some embodiments, the NSE modulates ATM proteinexpression. In some embodiments, the polynucleic acid polymer ismodified at a nucleoside moiety, at a phosphate moiety, at a 5′terminus, at a 3′ terminus, or a combination thereof. In someembodiments, the polynucleic acid polymer comprises an artificialnucleotide. In some embodiments, the artificial nucleotide is selectedfrom the group consisting of 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE),2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl(2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMA0E),2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), 2′-O-N-methylacetamido (2′-O-NMA), a locked nucleic acid(LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a1′,5′-anhydrohexitol nucleic acid (HNA), a morpholino, amethylphosphonate nucleotide, a thiolphosphonate nucleotide, and a2′-fluoro N3-P5′-phosphoramidite.

Disclosed herein, in certain embodiments, is a method of modulatingprotein expression comprising: (a) contacting an isolated polynucleicacid polymer to a target cell of a subject; (b) hybridizing thecontacted polynucleic acid polymer to a target motif either upstream ordownstream of a transposed element, wherein a hybridization of thecontacted polynucleic acid polymer to the target motif promotes orrepresses activation of a non-sense mediated RNA decay switch exon(NSE); (c) processing a mRNA transcript of the pre-processed mRNAtranscript, wherein the NSE is either present or absent in the mRNAtranscript; and (d) translating the processed mRNA transcript of stepc), wherein the presence or absence of the NSE modulates proteinexpression. In some embodiments, the protein is expressed from theprocessed mRNA transcript. In some embodiments, the presence of the NSEdownregulates protein expression. In some embodiments, the absence ofthe NSE upregulates protein expression. In some embodiments, thetransposed element is Alu or MER51. In some embodiments, the isolatedpolynucleic acid polymer comprises a sequence with at least 70%, 75%,80%, 85%, 90%, 95%, or 99% sequence identity to a sequence selected fromthe group consisting of SEQ ID NOs: 18-52. In some embodiments, thepolynucleic acid polymer is from about 10 to about 50 nucleotides inlength. In some embodiments, the isolated polynucleic acid polymerhybridizes to a target motif within Alu. In some embodiments, theisolated polynucleic acid polymer hybridizes to a target motif that iseither upstream or downstream of Alu. In some embodiments, the isolatedpolynucleic acid polymer hybridizes to a target motif downstream ofMER51. In some embodiments, activation of the NSE further induces exonskipping. In some embodiments, the NSE is located in intron 28. In someembodiments, the NSE modulates ATM protein expression. In someembodiments, the polynucleic acid polymer is modified at a nucleosidemoiety, at a phosphate moiety, at a 5′ terminus, at a 3′ terminus, or acombination thereof. In some embodiments, the polynucleic acid polymercomprises an artificial nucleotide. In some embodiments, the artificialnucleotide is selected from the group consisting of 2′-O-methyl,2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy,T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), 2′-O-N-methylacetamido(2′-O-NMA), a locked nucleic acid (LNA), an ethylene nucleic acid (ENA),a peptide nucleic acid (PNA), a 1′,5′-anhydrohexitol nucleic acid (HNA),a morpholino, a methylphosphonate nucleotide, a thiolphosphonatenucleotide, and a 2′-fluoro N3-P5′-phosphoramidite.

Disclosed herein, in certain embodiments, is a method of treating orpreventing a disease or condition associated with deregulation of ATMexpression in a subject in need thereof, the method comprising:administering to the subject a pharmaceutical composition comprising:(i) a non-sense mediated RNA decay switch exon (NSE)-activator agentthat interacts with a pre-processed mRNA transcript to promote inclusionof a NSE into a processed mRNA transcript; and (ii) a pharmaceuticallyacceptable excipient and/or a delivery vehicle; wherein the disease orcondition associated with deregulation of ATM expression is treated orprevented in the subject by the administration of the NSE-activatoragent. In some embodiments, the NSE-activator agent is an isolatedpolynucleic acid polymer. In some embodiments, the NSE-repressor agentis an isolated polynucleic acid polymer. In some embodiments, thepolynucleic acid polymer hybridizes to a motif within ATM intron 28. Insome embodiments, the polynucleic acid polymer hybridizes to a splicingregulatory motif that competes with the NSE for spliceosomal components.In some embodiments, the splicing regulatory motif comprises a crypticsplice site or a pseudoexon. In some embodiments, the pseudoexon is a 24nucleotide pseudoexon located at 3′ of a NSE in ATM intron 28 of thepre-mRNA transcript. In some embodiments, the polynucleic acid polymerhybridizes to a U2AF65 binding site. In some embodiments, thepolynucleic acid polymer hybridizes to a motif within a transposedelement, upstream of a transposed element, or downstream of a transposedelement. In some embodiments, the transposed element is Alu or MER51. Insome embodiments, the isolated polynucleic acid polymer hybridizes to atarget motif within Alu. In some embodiments, the isolated polynucleicacid polymer hybridizes to a target motif that is either upstream ordownstream of Alu. In some embodiments, the isolated polynucleic acidpolymer hybridizes to a target motif downstream of MER51. In someembodiments, the polynucleic acid polymer is from about 10 to about 50nucleotides in length. In some embodiments, the isolated polynucleicacid polymer comprises a sequence with at least 70%, 75%, 80%, 85%, 90%,95%, or 99% sequence identity to a sequence selected from the groupconsisting of SEQ ID NOs: 18-52. In some embodiments, the disease orcondition is cancer. In some embodiments, the polynucleic acid polymeris modified at a nucleoside moiety, at a phosphate moiety, at a 5′terminus, at a 3′ terminus, or a combination thereof. In someembodiments, the polynucleic acid polymer comprises an artificialnucleotide. In some embodiments, the artificial nucleotide is selectedfrom the group consisting of 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE),2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl(2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethyl aminoethyloxyethyl(2′-O-DMAEOE), 2′-O—N-methylacetamido (2′-O—NMA), a locked nucleic acid(LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a1′,5′-anhydrohexitol nucleic acid (HNA), a morpholino, amethylphosphonate nucleotide, a thiolphosphonate nucleotide, and a2′-fluoro N3-P5′-phosphoramidite. In some embodiments, the deliveryvehicle comprises a nanoparticle-based delivery vehicle.

Disclosed herein, in certain embodiments, is a method of treating orpreventing a disease or condition associated with a functional-ATMprotein deficiency in a subject in need thereof, the method comprising:administering to the subject a pharmaceutical composition comprising:(i) a non-sense mediated RNA decay switch exon (NSE)-repressor agentthat interacts with a pre-processed mRNA transcript to promote exclusionof a NSE into a processed mRNA transcript; and (ii) a pharmaceuticallyacceptable excipient and/or a delivery vehicle; wherein the disease orcondition associated with a functional-ATM protein deficiency is treatedor prevented in the subject by the administration of the NSE-repressoragent. In some embodiments, the NSE-activator agent is an isolatedpolynucleic acid polymer. In some embodiments, the NSE-repressor agentis an isolated polynucleic acid polymer. In some embodiments, thepolynucleic acid polymer hybridizes to a motif within ATM intron 28. Insome embodiments, the polynucleic acid polymer hybridizes to a splicingregulatory motif that competes with the NSE for spliceosomal components.In some embodiments, the splicing regulatory motif comprises a crypticsplice site or a pseudoexon. In some embodiments, the pseudoexon is a 24nucleotide pseudoexon located at 3′ of a NSE in ATM intron 28 of thepre-mRNA transcript. In some embodiments, the polynucleic acid polymerhybridizes to a U2AF65 binding site. In some embodiments, thepolynucleic acid polymer hybridizes to a motif within a transposedelement, upstream of a transposed element, or downstream of a transposedelement. In some embodiments, the transposed element is Alu or MER51. Insome embodiments, the isolated polynucleic acid polymer hybridizes to atarget motif within Alu. In some embodiments, the isolated polynucleicacid polymer hybridizes to a target motif that is either upstream ordownstream of Alu. In some embodiments, the isolated polynucleic acidpolymer hybridizes to a target motif downstream of MER51. In someembodiments, the polynucleic acid polymer is from about 10 to about 50nucleotides in length. In some embodiments, the isolated polynucleicacid polymer comprises a sequence with at least 70%, 75%, 80%, 85%, 90%,95%, or 99% sequence identity to a sequence selected from the groupconsisting of SEQ ID NOs: 18-52. In some embodiments, the disease orcondition is cancer. In some embodiments, the polynucleic acid polymeris modified at a nucleoside moiety, at a phosphate moiety, at a 5′terminus, at a 3′ terminus, or a combination thereof. In someembodiments, the polynucleic acid polymer comprises an artificialnucleotide. In some embodiments, the artificial nucleotide is selectedfrom the group consisting of 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE),2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl(2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethyl aminoethyloxyethyl(2′-O-DMAEOE), 2′-O-N-methylacetamido (2′-O-NMA), a locked nucleic acid(LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a1′,5′-anhydrohexitol nucleic acid (HNA), a morpholino, amethylphosphonate nucleotide, a thiolphosphonate nucleotide, and a2′-fluoro N3-P5′-phosphoramidite. In some embodiments, the deliveryvehicle comprises a nanoparticle-based delivery vehicle.

Disclosed herein, in certain embodiments, is a method of treating orpreventing a disease or condition in a subject in need thereof, themethod comprising: administering to the subject a pharmaceuticalcomposition comprising: (i) a non-sense mediated RNA decay switch exon(NSE)-activator agent that interacts with a pre-processed mRNAtranscript to promote inclusion of NSE into a processed mRNA transcript;and (ii) a pharmaceutically acceptable excipient and/or a deliveryvehicle; wherein the disease or condition is treated or prevented in thesubject by the administration of the NSE-activator agent. In someembodiments, the NSE-activator agent is an isolated polynucleic acidpolymer. In some embodiments, the NSE-repressor agent is an isolatedpolynucleic acid polymer. In some embodiments, the polynucleic acidpolymer hybridizes to a motif within ATM intron 28. In some embodiments,the polynucleic acid polymer hybridizes to a splicing regulatory motifthat competes with the NSE for spliceosomal components. In someembodiments, the splicing regulatory motif comprises a cryptic splicesite or a pseudoexon. In some embodiments, the pseudoexon is a 24nucleotide pseudoexon located at 3′ of a NSE in ATM intron 28 of thepre-mRNA transcript. In some embodiments, the polynucleic acid polymerhybridizes to a U2AF65 binding site. In some embodiments, thepolynucleic acid polymer hybridizes to a motif within a transposedelement, upstream of a transposed element, or downstream of a transposedelement. In some embodiments, the transposed element is Alu or MER51. Insome embodiments, the isolated polynucleic acid polymer hybridizes to atarget motif within Alu. In some embodiments, the isolated polynucleicacid polymer hybridizes to a target motif that is either upstream ordownstream of Alu. In some embodiments, the isolated polynucleic acidpolymer hybridizes to a target motif downstream of MER51. In someembodiments, the polynucleic acid polymer is from about 10 to about 50nucleotides in length. In some embodiments, the isolated polynucleicacid polymer comprises a sequence with at least 70%, 75%, 80%, 85%, 90%,95%, or 99% sequence identity to a sequence selected from the groupconsisting of SEQ ID NOs: 18-52. In some embodiments, the disease orcondition is cancer. In some embodiments, the disease or condition is adisease or condition associated with deregulation of ATM expression. Insome embodiments, the disease or condition is a disease or conditionassociated with a functional-ATM protein deficiency. In someembodiments, the polynucleic acid polymer is modified at a nucleosidemoiety, at a phosphate moiety, at a 5′ terminus, at a 3′ terminus, or acombination thereof. In some embodiments, the polynucleic acid polymercomprises an artificial nucleotide. In some embodiments, the artificialnucleotide is selected from the group consisting of 2′-O-methyl,2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy,T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMA0E), 2′-O-dimethylaminopropyl (2′-O-DMAP),T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), 2′-O-N-methylacetamido(2′-O-NMA), a locked nucleic acid (LNA), an ethylene nucleic acid (ENA),a peptide nucleic acid (PNA), a 1′,5′-anhydrohexitol nucleic acid (HNA),a morpholino, a methylphosphonate nucleotide, a thiolphosphonatenucleotide, and a 2′-fluoro N3-P5′-phosphoramidite. In some embodiments,the delivery vehicle comprises a nanoparticle-based delivery vehicle.

Disclosed herein, in certain embodiments, is a method of treating orpreventing a disease or condition in a subject in need thereof, themethod comprising: administering to the subject a pharmaceuticalcomposition comprising: (i) a non-sense mediated RNA decay switch exon(NSE)-repressor agent that interacts with a pre-processed mRNAtranscript to promote exclusion of an NSE into a processed mRNAtranscript; and (ii) a pharmaceutically acceptable excipient and/or adelivery vehicle; wherein the disease or condition is treated orprevented in the subject by the administration of the NSE-repressoragent. In some embodiments, the NSE-activator agent is an isolatedpolynucleic acid polymer. In some embodiments, the NSE-repressor agentis an isolated polynucleic acid polymer. In some embodiments, thepolynucleic acid polymer hybridizes to a motif within ATM intron 28. Insome embodiments, the polynucleic acid polymer hybridizes to a splicingregulatory motif that competes with the NSE for spliceosomal components.In some embodiments, the splicing regulatory motif comprises a crypticsplice site or a pseudoexon. In some embodiments, the pseudoexon is a 24nucleotide pseudoexon located at 3′ of a NSE in ATM intron 28 of thepre-mRNA transcript. In some embodiments, the polynucleic acid polymerhybridizes to a U2AF65 binding site. In some embodiments, thepolynucleic acid polymer hybridizes to a motif within a transposedelement, upstream of a transposed element, or downstream of a transposedelement. In some embodiments, the transposed element is Alu or MER51. Insome embodiments, the isolated polynucleic acid polymer hybridizes to atarget motif within Alu. In some embodiments, the isolated polynucleicacid polymer hybridizes to a target motif that is either upstream ordownstream of Alu. In some embodiments, the isolated polynucleic acidpolymer hybridizes to a target motif downstream of MER51. In someembodiments, the polynucleic acid polymer is from about 10 to about 50nucleotides in length. In some embodiments, the isolated polynucleicacid polymer comprises a sequence with at least 70%, 75%, 80%, 85%, 90%,95%, or 99% sequence identity to a sequence selected from the groupconsisting of SEQ ID NOs: 18-52. In some embodiments, the disease orcondition is cancer. In some embodiments, the disease or condition is adisease or condition associated with deregulation of ATM expression. Insome embodiments, the disease or condition is a disease or conditionassociated with a functional-ATM protein deficiency. In someembodiments, the polynucleic acid polymer is modified at a nucleosidemoiety, at a phosphate moiety, at a 5′ terminus, at a 3′ terminus, or acombination thereof. In some embodiments, the polynucleic acid polymercomprises an artificial nucleotide. In some embodiments, the artificialnucleotide is selected from the group consisting of 2′-O-methyl,2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy,T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMA0E), 2′-O-dimethylaminopropyl (2′-O-DMAP),T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), 2′-O-N-methylacetamido(2′-O-NMA), a locked nucleic acid (LNA), an ethylene nucleic acid (ENA),a peptide nucleic acid (PNA), a 1′,5′-anhydrohexitol nucleic acid (HNA),a morpholino, a methylphosphonate nucleotide, a thiolphosphonatenucleotide, and a 2′-fluoro N3-P5′-phosphoramidite. In some embodiments,the delivery vehicle comprises a nanoparticle-based delivery vehicle.

Disclosed herein, in certain embodiments, is a method of modulatingprotein expression comprising: (a) contacting an isolated polynucleicacid polymer to a target cell of a subject; (b) hybridizing thecontacted polynucleic acid polymer to a target motif within a transposedelement, wherein a hybridization of the contacted polynucleic acidpolymer to the target motif either promotes or represses activation ofan alternative splice site; (c) processing a mRNA transcript of thepre-processed mRNA transcript, wherein the alternative splice site iseither present or absent in the mRNA transcript; and (d) translating theprocessed mRNA transcript of step c), wherein the presence or absence ofthe alternative splice site modulates protein expression. In someembodiments, the transposon element is on the pre-processed mRNAtranscript.

Disclosed herein, in certain embodiments, is a method of modulatingprotein expression comprising: (a) contacting an isolated polynucleicacid polymer to a target cell of a subject; (b) hybridizing thecontacted polynucleic acid polymer to a target motif either upstream ordownstream of a transposed element, wherein a hybridization of thecontacted polynucleic acid polymer to the target motif promotes orrepresses activation of an alternative splice site; (c) processing amRNA transcript of the pre-processed mRNA transcript, wherein thealternative splice site is either present or absent in the mRNAtranscript; and (d) translating the processed mRNA transcript of stepc), wherein the presence or absence of the alternative splice sitemodulates protein expression. In some embodiments, the transposonelement is on the pre-processed mRNA transcript.

Disclosed herein, in certain embodiments, is a method of modulatingprotein expression comprising: (a) contacting an isolated polynucleicacid polymer to a target cell of a subject; (b) hybridizing thecontacted polynucleic acid polymer to a target motif on a pre-processedmRNA transcript, wherein hybridization of the contacted polynucleic acidpolymer to the target motif either promotes or represses activation ofan alternative splice site; (c) processing a mRNA transcript of thepre-processed mRNA transcript, wherein the alternative splice site iseither present or absent in the mRNA transcript; and (d) translating theprocessed mRNA transcript of step c), wherein the presence or absence ofthe alternative splice site modulates protein expression. In someembodiments, the protein is expressed from the processed mRNAtranscript. In some embodiments, the presence of NSE downregulatesprotein expression. In some embodiments, the absence of NSE upregulatesprotein expression. In some embodiments, the polynucleic acid polymerhybridizes to a motif within ATM intron 28. In some embodiments, themotif is a splicing regulatory motif that competes with NSE for aspliceosomal component. In some embodiments, the splicing regulatorymotif comprises a cryptic splice site or a pseudoexon. In someembodiments, the pseudoexon is a 24 nucleotide pseudoexon located at 3′of a NSE in ATM intron 28 of the pre-mRNA transcript. In someembodiments, the motif is a U2AF65 binding site. In some embodiments,the motif is a motif within a transposed element, upstream of atransposed element, or downstream of a transposed element. In someembodiments, the transposed element is Alu or MER51. In someembodiments, the isolated polynucleic acid polymer hybridizes to atarget motif within Alu. In some embodiments, the isolated polynucleicacid polymer hybridizes to a target motif that is either upstream ordownstream of Alu. In some embodiments, the isolated polynucleic acidpolymer hybridizes to a target motif downstream of MER51. In someembodiments, the polynucleic acid polymer is from about 10 to about 50nucleotides in length. In some embodiments, the isolated polynucleicacid polymer comprises a sequence with at least 70%, 75%, 80%, 85%, 90%,95%, or 99% sequence identity to a sequence selected from the groupconsisting of SEQ ID NOs: 18-52.

Disclosed herein, in certain embodiments, is a pharmaceuticalcomposition comprising: (a) a non-sense mediated RNA decay switch exon(NSE)-activator agent that interacts with a pre-processed mRNAtranscript to promote inclusion of NSE into a processed mRNA transcript,or a non-sense mediated RNA decay switch exon (NSE)-repressor agent thatinteracts with a pre-processed mRNA transcript to promote exclusion ofan NSE into a processed mRNA transcript; and (b) a pharmaceuticallyacceptable excipient and/or a delivery vehicle. In some embodiments, theNSE-activator agent is an isolated polynucleic acid polymer. In someembodiments, the NSE-repressor agent is an isolated polynucleic acidpolymer. In some embodiments, the polynucleic acid polymer hybridizes toa motif within ATM intron 28. In some embodiments, the polynucleic acidpolymer hybridizes to a splicing regulatory motif that competes with theNSE for a spliceosomal component. In some embodiments, the splicingregulatory motif comprises a cryptic splice site or a pseudoexon. Insome embodiments, the pseudoexon is a 24 nucleotide pseudoexon locatedat 3′ of NSE in ATM intron 28 of the pre-mRNA transcript. In someembodiments, the polynucleic acid polymer hybridizes to a U2AF65 bindingsite. In some embodiments, the polynucleic acid polymer hybridizes to amotif within a transposed element, upstream of a transposed element, ordownstream of a transposed element. In some embodiments, the transposedelement is Alu or MER51. In some embodiments, the isolated polynucleicacid polymer hybridizes to a target motif within Alu. In someembodiments, the isolated polynucleic acid polymer hybridizes to atarget motif that is either upstream or downstream of Alu. In someembodiments, the isolated polynucleic acid polymer hybridizes to atarget motif downstream of MER51. In some embodiments, the polynucleicacid polymer is from about 10 to about 50 nucleotides in length. In someembodiments, the isolated polynucleic acid polymer comprises a sequencewith at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity toa sequence selected from the group consisting of SEQ ID NOs: 18-52.

Disclosed herein, in certain embodiments, is a cell comprising apharmaceutical composition comprising: (a) a non-sense mediated RNAdecay switch exon (NSE)-activator agent that interacts with apre-processed mRNA transcript to promote inclusion of NSE into aprocessed mRNA transcript, or a non-sense mediated RNA decay switch exon(NSE)-repressor agent that interacts with a pre-processed mRNAtranscript to promote exclusion of an NSE into a processed mRNAtranscript; and (b) a pharmaceutically acceptable excipient and/or adelivery vehicle. In some embodiments, the NSE-activator agent is anisolated polynucleic acid polymer. In some embodiments, theNSE-repressor agent is an isolated polynucleic acid polymer. In someembodiments, the polynucleic acid polymer hybridizes to a motif withinATM intron 28. In some embodiments, the polynucleic acid polymerhybridizes to a splicing regulatory motif that competes with the NSE fora spliceosomal component. In some embodiments, the splicing regulatorymotif comprises a cryptic splice site or a pseudoexon. In someembodiments, the pseudoexon is a 24 nucleotide pseudoexon located at 3′of NSE in ATM intron 28 of the pre-mRNA transcript. In some embodiments,the polynucleic acid polymer hybridizes to a U2AF65 binding site. Insome embodiments, the polynucleic acid polymer hybridizes to a motifwithin a transposed element, upstream of a transposed element, ordownstream of a transposed element. In some embodiments, the transposedelement is Alu or MER51. In some embodiments, the isolated polynucleicacid polymer hybridizes to a target motif within Alu. In someembodiments, the isolated polynucleic acid polymer hybridizes to atarget motif that is either upstream or downstream of Alu. In someembodiments, the isolated polynucleic acid polymer hybridizes to atarget motif downstream of MER51. In some embodiments, the polynucleicacid polymer is from about 10 to about 50 nucleotides in length. In someembodiments, the isolated polynucleic acid polymer comprises a sequencewith at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity toa sequence selected from the group consisting of SEQ ID NOs: 18-52.

Disclosed herein, in certain embodiments, is a method, use, composition,vector, or agent substantially described herein, optionally withreference to the accompanying figures.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-FIG. 1C illustrate an identification of a U2AF-repressed crypticexon in ATM intron 28. FIG. 1A shows a schematics of the cryptic exon(termed here NSE for NMD-switch exon) activation. NSE sequence (SEQ IDNO: 62) (upper panel) is boxed, asterisk denotes rs609261, and blackrectangles show the indicated antisense oligonucleotides. Genome browserviews of RNA-Seq data from RNAi- or SSO-mediated depletions of bothU2AF35 isoforms (ab-), U2AF35a (a-), U2AF35b (b-) and controls (c) areshown in the lower panel. SSOs targeting 3′ ss of U2AF1 exons Ab and 3and U2AF35 siRNA were as previously described. Y axis, read densities.NSE inclusion/exclusion is schematically shown by dotted lines at thetop. ATM exons (gray boxes) are numbered. The 29-nt NS E introduced astop codon in the ATM mRNA. FIG. 1B shows validation of the NSEactivation by RT-PCR (upper panel) in independent depletions (lowerpanel). RT-PCR primers (ATM-F, ATM-R, FIG. 20) are denoted by arrows inpanel A. Spliced products are shown to the right, the percentage oftranscripts with NSE is at the top. Error bars denote SDs of twotransfections experiments (***, p<0.0001, **, p<0.001). FIG. 1C showsNSE inclusion in mature transcripts inversely correlates with residualU2AF (r =Pearson correlation). Estimates of heterodimer levels weredetermined.

FIG. 2A-FIG. 21 show NSE activation and ATM expression modified byrs609261. Allelic frequencies at rs609261 are shown in the indicatedpopulations (FIG. 2A). FIG. 2B shows exemplary minigene schematics. AnXhoI/XbaI segment of ATM containing NSE and exon 29 was cloned betweenU2AF1 exons 2 and 4 (black boxes). RT-PCR primers to amplify exogenoustranscripts (PL3 and ATM-R, FIG. 20) are denoted by arrows. FIG. 2Cshows the rs609261-dependent NSE activation in exogenous pre-mRNAs.HEK293 cells depleted of U2AF35 or U2AF65 were transiently transfectedwith T (black) and C (grey) minigenes. Final concentration of the U2AF35and U2AF65 siRNAs was 30 and 60 nM, respectively. FIG. 2D illustratesthe identification of cell lines homozygous at rs609261 (asterisk). NSEis boxed. FIG. 2D discloses SEQ ID NOS 63 and 64, respectively, in orderof appearance. FIG. 2E and FIG. 2F show allele-specific activation ofNSE in endogenous transcripts limits ATM expression in a dose-dependentmanner. The source of endogenous transcripts is at the bottom,antibodies are to the right Concentration of siRNAs in cultures was 3,10 and 30 nM. C1, C2, control siRNAs. Transfection efficiency wasmonitored by a GFP-plasmid and fluorescent microscopy. FIG. 2G shows UPF1 depletion increased NSE activation (upper panel) and upregulatedisoform U2AF1c (lower panel). The U2AF1c isoform contains both exons Aband 3 and is repressed by NMD. Final concentration of the UPF1 siRNA was7, 20 and 60 nM (SC=a scrambled control). Error bars are SDs ofindependent transfections. FIG. 2H shows NSE inclusion levels in cellsdepleted of U2AF-related proteins and a subset of heterogeneous nuclearRNPs. Error bars denote SDs of two transfections. Immunoblots are shownto the right Final concentration of the U2AF35 siRNA was 25 nM; theremaining siRNAs were at 60 nM (C=controls). FIG. 21 showsoverexpression of PUF60 induced NSE skipping. Immunoblots are shownbelow, antibodies to the right.

FIG. 3A-FIG. 3D illustrate rescue of U2AF-repressed ATM expression bySSOs targeting NSE. FIG. 3A and FIG. 3B show efficient SSO-mediated NSEinhibition in exogenous (FIG. 3A) and endogenous (FIG. 3B) ATMtranscripts. Mean NSE inclusion levels of two transfection experimentsare shown in the right panels. FIG. 3C shows restoration of ATM proteinlevels by SSOs that blocks access to NSE. Cells lacking U2AF35 andcontrol cells were transfected with the SSO targeting the NSE 3′ss and acontrol SSOs (FIG. 1A and FIG. 20). After 48 hrs, the cells were exposedto ionizing radiation (IR, 10 Gy) and harvested 1 hr later. Cell lysateswere separated using a gradient SDS-PAGE. Western blotting was withantibodies shown to the right FIG. 3D shows dose-dependentreconstitution of ATM expression SSO—NSE3 in depleted cells.

FIG. 4A-FIG. 4H show identification of intronic cis-elements and SSOsthat modulate NSE activation. FIG. 4A shows schematics of twopseudoexons in ATM intron 28. Canonical exons (numbered) are shown asgrey boxes, NSE as a white box, and PE as a checkered box. Asteriskindicates location of the IVS28-159A>G substitution, causing A-T. Inthis A-T case, both NSE and PE were included in the ATM mRNA togetherwith the intervening sequence because NSE is separated from PE by lessthan the minimal size of human intron. Canonical and aberranttranscripts are denoted by dotted lines above and below the pre-mRNA,respectively. Middle panel shows RNA-Seq read densities for NSE in cellsdepleted of both U2AF35 isoforms (ab-) together with U2AF65tags/high-confidence binding sites (horizontal lines/rectangles)identified by crosslinking and immunoprecipitation. The 100 basewisevertebrate conservation by Phylop (100 VC) is shown at the bottom. Lowerpanel shows mutations (in red and underlined) introduced in theC-minigene. (SEQ ID NOS 65-67, respectively, in order of appearance).FIG. 4B shows splicing pattern of wildtype and mutated C minigenes.Mutations are shown in panel A; RNA products are shown schematically tothe right. The largest product produced by clone PE de1PPT/AG containsthe shortened pseudointron (42 nt). FIG. 4C shows splicing pattern of Cminigenes mutated in NSE (lanes 2, 3, 7 and 8) or PE (lanes 4, 5, 9 and10) in (mock) depleted HEK293 cells. Mutations are at the bottom andminigene sequences in FIG. 21. Spliced products are schematically shownto the right; a hairpin symbol above PE denotes the MIR stem-loopinsertion. FIG. 4D and FIG. 4E illustrate SSO-induced pseudoexonswitching. Transfected minigenes are shown at the top, spliced productsto the right and SSOs at the bottom. SSO sequences are in FIG. 20. Finalconcentration of SSOs shown in panels D-G was 3, 10 and 30 nM. FIG. 4Fshows SSOs targeting PE induced NSE skipping. FIG. 4G shows SSOstargeting a sequence activating NSE upon deletion (PEde1PPT/AG; panel Aand B) inhibit PE. FIG. 4H shows NSE activation is haplotype-dependentMinigene haplotypes at the indicated variants are shown at the bottom.Columns represent mean NSE inclusion, error bars are SDs, and asterisksdenote statistically significant differences as in FIG. 1B.

FIG. 5A-FIG. 5G show exon-centric regulation of ATM signaling. FIG. 5Ashows U2AF-regulated gene- and exon-level expression changes inMRN-ATM-CHEK2-CDC25-cdc2/cyclin B pathway (left panel). Log2fold- andq-values are shown in parentheses. Exon usage of CHEK2 and CDC25A genesis shown by RNA-Seq browser shots; PCR validation gels are in the rightpanels. CHEK2 exon 9 is a NMD switch exon; exon 11 encodes a portion ofthe kinase domain. Full spectrum of U2AF-mediated expression changes inthe ATM signaling pathway is shown in FIG. 9; examples of theU2AF-mediated splicing regulation are in FIG. S3-S6. FIG. 5B showsimpaired ATM signaling in U2AF35 depleted cells following IR. HEK293cells were (mock) depleted of U2AF35 and subjected to IR (10 Gy) 48 hrslater. Expression was examined by immunoblotting at the indicated timepoints. Antibodies are shown to the right CHEK2 exon 9 skipping levelsare at the bottom; their measurements in control (U2AF35+) and depletedcells (U2AF35-) are in panel FIG. 5C. FIG. 5D shows CHEK2 exon 9inclusion in UPF1 depleted cells. Final concentration of the UPF1 siRNA(FIG. 20) was 12.5, 25, 50, and 100 nM. FIG. 5E shows repression ofCHEK2 exon 9 by SSO reduced CHEK2 levels and promoted NSE inclusion.Final concentration of SSO targeting CHEK2 exon 9 was 3, 10 and 30 nM.FIG. 5F shows CHEK2 exon 9 inclusion upon transfection of HEK293 cellswith the indicated SSOs. FIG. 5G shows a lack of SF3B1 induced CHEK2exon 9 skipping but did not alter NSE activation. Final concentration ofeach siRNA targeting SF3B 1 was 20 nM.

FIG. 6 shows rescue of NSE repression by cancer-associated mutations inU2AF35. Rescue of U2AF35-dependent NSE splicing of the C minigene byzinc finger 1 and 2 substitutions in U2AF35 (upper panel). Allsubstitutions were made in the U2AF1a construct (35a). Cancer-associatedmutations (bottom) are boxed; splice products are to the rightImmunoblot with U2AF35 and GFP antibodies is shown in the lower panel(ex=exogenous; en=endogenous U2AF35).

FIG. 7 shows SSO-based modulation of gene expression by pseudoexontargeting. Canonical exons are shown as grey boxes, a nonsense-mediatedRNA decay (NMD) switch exon as a black box, pseudoexons as white boxes.Canonical splicing is shown by dotted lines. Pseudosplice sitescompeting with the NMD exon are shown below the RNA precursor. SSOactivators/repressors are denoted by horizontal black/grey bars,respectively. Splicing regulatory motifs or secondary structures thatcompete with NMD switch exons for spliceosome components such as U2AF,heterogeneous nuclear ribonucleoproteins, or serine/arginine-richproteins, for inclusion to mature transcripts are not shown forsimplicity. They can be predicted by computational methods described indetails previously (for example, Kralovicova, J. and Vorechovsky, I.(2007) Global control of aberrant splice site activation by auxiliarysplicing sequences: evidence for a gradient in exon and introndefinition. Nucleic Acids Res., 35, 6399-6413, and references therein)or determined by experimental methods , including RNA crosslinking andimmunoprecipitation, mutagenesis of splicing substrates and RNA foldingstudies.

FIG. 8A-FIG. 8C show SSO-mediated NSE repression enhances ATMexpression. FIG. 8A shows SSO—NSE3 increased expression of total andactivated ATM. HEK293 cells were (mock)-depleted of U2A F35,cotransfected with X press-tagged CHEK2 and SSO NSE3/control (SSO-C),exposed to ionizing radiation (IR) and harvested 30 minutes later. Celllysates were immunoblotted with the indicated antibodies. Finalconcentration of siRNA and SSOs was 30 nM. The amount of plasmidsexpressing CHEK2 was 30, 90 and 270 ng; DNA from the empty vector wasadded to a final concentration of 270 ng/mL. Ex/enCHEK2, signal fromexogenous and endogenous CHEK2, as detected by the D9C6 antibody. FIG.8B and FIG. 8C show increased expression of exogenous CHEK2 by an SSOtargeting NMD switch exon 9 (SSO CHEK2). Constant amounts of SSO CHEK2were cotransfected with increasing amounts of Xpress-CHEK2 and constantamounts of GFP plasmids as transfection and loading control (B) and viceversa (C). Antibodies are to the right.

FIG. 9 illustrates an exemplary map of U2AF-regulated functional ATMinteractions. U2AF-regulated ATM signaling network is highlighted by redarrows/pink background. Genes up-/down-regulated in cells depleted ofU2AF35 are shown in red/dark green, respectively. Genes exhibitingsignificantly altered exon usage are shown in yellow. The ATM signalingmap shows ATM-interacting proteins (purple)/protein complexes (lightgreen). Arrows correspond to activation, T-shaped edges to inhibitionand open circles denote unknown regulations. Containment links are shownas green edges.

FIG. 10A-FIG. 0B show exon usage in CDC25B and CDC25C in cells depletedof U2AF35. Genomic browser views of RNA-Seq data in control (ctr) anddepleted (ab-) cells (left panels in FIG. 10A and FIG. 10B). PCR primersare shown by arrows, differentially used exons are denoted by blackrectangles. RefSeq exon annotation is shown at the bottom. Validation ofRNA-Seq data using RT-PCR with RNA extracted from cells depleted of eachU2AF subunits and U2AF-related proteins (right panels in FIG. 10A andFIG. 10B).

FIG. 11A-FIG. 11C shows U2AF-regulated exon usage in TTK, PINland CDK1.Genomic browser views of RNA-Seq data in control (ctr) and depleted(ab-) cells (in FIG. 11A, left panel of FIG. 11B, and FIG. 11C). PCRprimers are shown by arrows, differentially used exons are denoted byblack rectangles. RefSeq exon annotation is shown at the bottom.Validation of RNA-Seq data using RT-PCR with RNA extracted from cellsdepleted of each U2AF subunits and U2AF-related proteins (right panel inFIG. 11B). FIG. 11A discloses SEQ ID NO: 127.

FIG. 12A-FIG. 12D show RNA processing of RAD50 and EZH2 in depletedcells. Genomic browser views of RNA-Seq data in control (ctr) anddepleted (ab-) cells (left panels in FIG. 12A and FIG. 12B and in FIG.12C and FIG. 12D). PCR primers are shown by arrows, differentially usedexons are denoted by black rectangles. RefSeq exon annotation is shownat the bottom. Validation of RNA-Seq data using RT-PCR with RNAextracted from cells depleted of each U2AF subunits and U2AF-relatedproteins (right panels in FIG. 12A and FIG. 12B).

FIG. 13A-FIG. 13B show U2AF35-controlled exon usage of thepeptidyl-prolyl isomerase PIN2 and components of the shelterin complex.

FIG. 14A-FIG. 14D show U2AF control of RARA fusion partners. FIG. 14Ddiscloses SEQ ID NO: 68.

FIG. 15A-FIG. 15E show NSE activation in normal tissue and leukemiccells. NSE inclusion levels were measured in 19 human tissues (FIG. 15A)and 17 AML/CMML bone marrow samples (FIG. 15B) using primers ATM-F andATM-R (FIG. 1, FIG. 20). Exon inclusion was quantified. Means werecompared with an unpaired t-test (FIG. 15C). FIG. 15D and FIG. 15E showinclusion levels of U2AF-repressed (FIG. 15D) and -activated (FIG. 15E)exons in lymphoblastoid cell lines (top). Cells were exposed to cold andheat shock at the indicated temperatures. ES, exon skipping; EI, exoninclusion.

FIG. 16A-FIG. 16B show identification of transposed elements in ATMintron 28 that influence NSE activation. FIG. 16A shows the location oftransposed elements in intron 28 and schematics of NSE activation.Canonical exons are shown as grey boxes, the NSE as a white box, intronsflanking the NSE as lines and their splicing by dotted lines. Transposedelements are shown as horizontal white rectangles below the primarytranscript; UC, a unique sequence lacking recognizable transposons.Their deletions are numbered 1-6, which corresponds to lane numbers inpanel B. RT PCR primers are denoted by black arrows. A scale is at thetop. The NSE sequence is boxed in the lower panel. Constructs lackingthe sense Alu (Alu+) repeatedly failed to ligate/propagate and were notexamined. FIG. 16A discloses SEQ ID NO: 69). FIG. 16B shows deletion ofantisense Alu and MER51 elements alters NSE activation. Wild-type (WT)and mutated constructs (designated 1-6) were transiently transfectedinto HEK293 cells (mock)depleted of U2AF35. NSE+/−, RNA productswith/without NSE. Columns represent mean NSE inclusion (%), error barsSDs of 2 transfection experiments. Asterisks denote two-tailed Pvalues<0.01 (t-test).

FIG. 17A-FIG. 17C show identification of intronic SSOs that activate orrepress NSE. FIG. 17A shows the location of tested SSOs in intron 28relative to transposed elements. For legend, see FIG. 16A. FIG. 17Bshows the identification of intron 28 SSOs that alter NSE activation inexogenous transcripts. Illustrative SSOs are listed in Table 2. The “x”symbol denotes multiple negative controls, dotted line the average NSEinclusion, error bars SDs of two transfections experiments. Columnsrepresent mean inclusion levels, asterisks show significant P values.FIG. 17C shows SSOs targeting single-stranded regions tended to repressendogenous NSE. r, Pearson correlation coefficient. The P value is inparentheses.

FIG. 18A-FIG. 18B show TMC-SA-assisted delivery of SSO—NSE3 to humancell lines leads to NSE repression. FIG. 18A shows NSE inclusion inHEK293 cells is inhibited upon exposure of SSO—NSE3/TMC-SAnanocomplexes. N/P ratio was 20, 40 and 80 (Sc=a scrambled control withthe same modification , M =size marker). Error bars denote SDs of twotransfections experiments. P values are shown at the top for theindicated comparisons. FIG. 18B shows NSE repression in VAVY cellsexposed to SSO—NSE3/TMC-SA complexes.

FIG. 19 shows inverted repeats in the MER51 consensus sequence (SEQ IDNO: 71) with ATM intron 28 (SEQ ID NO: 70) (v, transversions; i,transitions). Most stable inverted repeats in the ATM MER51A areunderlined and highlighted; purine-rich single-stranded regions are inred; the long terminal repeat homology originally described for theMER51 family is in italics. The aligned segment corresponds to deletion4 shown in FIG. 16a . The MER51A consensus sequence is in the antisenseorientation.

FIG. 20 illustrates exemplary synthetic DNA and RNA sequences. (SEQ IDNOS 72-114, respectively, in order of appearance).

FIG. 21 shows exemplary sequences of splicing reporter constructsmutated in NSE and PE. (SEQ ID NOS 115-119, respectively, in order ofappearance).

FIG. 22 shows auxiliary splicing elements in NSE and PE.

FIG. 23 shows a summary of U2AF35-regulated transcripts involved in NMD.

DETAILED DESCRIPTION OF THE INVENTION

Intervening sequences or introns are removed by a large and highlydynamic RNA-protein complex termed the spliceosome, which orchestratescomplex interactions between primary transcripts, small nuclear RNAs(snRNAs) and a large number of proteins. Spliceosomes assemble ad hoc oneach intron in an ordered manner, starting with recognition of the 5′splice site (5′ ss) by U1 snRNA or the 3′ss by the U2 pathway, whichinvolves binding of the U2 auxiliary factor (U2AF) to the 3′ss region tofacilitate U2 binding to the branch point sequence (BPS). U2AF is astable heterodimer composed of a U2AF2-encoded 65-kD subunit (U2AF65),which binds the polypyrimidine tract (PPT), and a U2AF1-encoded 35-kDsubunit (U2AF35), which interacts with highly conserved AG dinucleotidesat 3′ss and stabilizes U2AF65 binding. In addition to the BPS/PPT unitand 3′ss/5′ss, accurate splicing requires auxiliary sequences orstructures that activate or repress splice site recognition, known asintronic or exonic splicing enhancers or silencers. These elements allowgenuine splice sites to be recognized among a vast excess of cryptic orpseudo-sites in the genome of higher eukaryotes, which have the samesequences but outnumber authentic sites by an order of magnitude.Although they often have a regulatory function, the exact mechanisms oftheir activation or repression are poorly understood.

Exome sequencing studies have revealed a highly restricted pattern ofsomatic mutations in U2AF1/U2AF2 and other genes involved in 3′ssrecognition (SF3B1, ZRSR2, SF1, SF3A1, PRPF40B, and SRSF2) in cancercells, most prominently myelodysplastic syndromes. These genes encodeproducts that often interact during spliceosome assembly, suggesting theexistence of shared pathways in oncogenesis, which is further supportedby a high degree of mutual exclusivity of cancer-associated mutations.Genome-wide transcriptome profiling in leukemic samples carrying thesemutations detected numerous alterations in splicing of mRNA precursors,however, key links between specific RNA processing defects and cancerinitiation or progression have remained obscure, despite the greatpromise of these targets for therapeutic modulation. Theinterconnections between these RNA-binding proteins and DNA damageresponse (DDR) pathways remain to be fully characterized.

Mutations in traditional (BPS/PPT/3′ss/5′ss) and auxiliary splicingmotifs often cause aberrant splicing, such as exon skipping or crypticexon or splice-site activation, and contribute significantly to humanmorbidity and mortality. Both aberrant and alternative splicing patternscan be influenced by natural DNA variants in exons and introns, whichplay an important role in heritability of both Mendelian and complextraits. However, the molecular mechanisms that translate the allele- orhaplotype-specific RNA expression to phenotypic variability as well asinteractions between intronic and exonic variant alleles andtrans-acting factors are largely obscure.

Antisense technology has now reached important clinical applications.For example, antisense splice-switching oligonucleotides (SSOs)targeting the ATM gene have been used to repair splicing mutations inataxia-telangiectasia (A-T) and were successful in normalizing ATMprotein levels (Du et al., 2011; Du et al., 2007).

A large fraction of both leukemias and solid tumors show deregulation ofATM expression (for example, Stankovic et al., 1999; Starczynski et al.,2003). Chemical inhibitors of ATM (wortmannin, CP-466722, KU-55933, andKU60019) have not reached clinical trials, largely because ofnonspecific effects and/or high toxicity, although KU-559403 has showngood bioavailability and reliably conferred radiosensitivity.

In some instances, the ability to up or down regulate gene expression ina sequence-specific manner is desirable.

In certain embodiments, provided herein is a method of screening asubject or a population of subjects for susceptibility to functional-ATMprotein deficiency, wherein the screening comprises determining thepresence of a non-thymine variant residue rs609261 located at position-3 relative to the 3′ splice site of NSE (cryptic or nonsense-mediatedRNA decay switch exon in ATM intron 28) of the human genome, wherein thepresence of a non-thymine variant residue rs609261 indicates that thesubject (or group of subjects) has, or is susceptible to, functional-ATMprotein deficiency.

The term “functional ATM-protein deficiency” means the reduction in thepresence/expression of ATM protein that is functional in a subject, cellor tissue. Functional ATM-deficiency is the result of a functionalvariant rs609261 in ATM intron 28 that alters RNA processing of ATMprecursor messenger RNA (pre-mRNA). Cytosine allele at rs609261 resultsin a higher inclusion of a nonsense-mediated RNA decay switch exon(termed here NSE) in ATM mRNA than a thymine allele at this position,limiting the expression of ATM protein more efficiently than the thymineallele. This limitation can be removed or modulated by novel SSOs thatblock access to NSE or to NSE-regulatory sequences in the same intron,leading to derepression or inhibition of ATM protein, respectively.

In some embodiments, provided herein is a method of selecting a subjector a population of subjects for treatment or prophylaxis, wherein thesubject is susceptible to functional-ATM protein deficiency, the methodcomprising determining the presence of a non-thymine variant residuers609261 located at position -3 relative to the 3′ splice site of NSE(cryptic exon in ATM intron 28) of the human genome, wherein thepresence of a non-thymine variant residue rs609261indicates that thesubject has, or is susceptible to, functional-ATM protein deficiency,and selecting such subject for treatment with an agent arranged toincrease functional-ATM levels in the subject.

According to another aspect of the invention, there is provided a methodof treatment or prevention of functional-ATM protein deficiency in asubject, the method comprising identifying the presence of a non-thyminevariant residue rs609261 located at position −3 relative to the 3′splice site of NSE of the human genome, wherein the presence of anon-thymine variant residue rs609261 indicates that the subject has, oris susceptible to, functional-ATM protein deficiency, and administrationof an agent to the subject, which is arranged to increase functional-ATMlevels.

According to another aspect of the invention, there is provided a methodof treatment or prevention of a condition associated with afunctional-ATM protein deficiency, comprising the administration of aNSE repressor agent arranged to increase levels of functional ATMprotein, wherein the agent is arranged to bind to a NSE in ATM intron 28of the pre-mRNA transcript or to NSE-activating regulatory sequences inthe same intron to decrease inclusion of the NSE in the maturetranscript.

According to another aspect of the invention, there is provided a methodof treatment or prevention of a condition associated with deregulationof ATM expression in a subject comprising the administration of aNSE-activator agent, wherein the NSE-activator agent is arranged toincrease NSE inclusion in the ATM mature RNA transcript by binding toNSE-inhibiting regulatory motifs in ATM intron 28.

NSE-inhibiting regulatory motifs in ATM intron 28 may comprise sequencesthat compete with NSE for spliceosomal components, such as a 24nucleotide pseudoexon (PE) located 3′ of NSE in ATM intron 28 of thepre-mRNA transcript or U2AF65 binding site upstream of the pseudoexon.

According to another aspect of the invention, there is provided a methodof treatment or prevention of cancer in a subject comprising theadministration of a NSE-activator agent arranged to increase a cancercell's susceptibility to DNA damaging agents that induce double strandDNA breaks, such as radiotherapy, wherein the NSE-activator agent isarranged to increase NSE inclusion in the ATM mature RNA by binding NSEregulatory motifs in ATM intron 28; and treating the subject with DNAdamaging agents that cause double strand breaks, such as radiotherapy orchemotherapy.

According to another aspect of the invention, there is provided a methodof increasing a cell's susceptibility to cytotoxic therapy, such asradiotherapy treatment, comprising the reduction of ATM proteinexpression by administration of a NSE-activator agent arranged toincrease NSE inclusion in ATM mature RNA transcript by binding toregulatory motifs in ATM intron 28.

The regulatory motifs in ATM intron 28 may compete with NSE forspliceosomal components, wherein such motifs may comprise a 24nucleotide pseudoexon (PE) located 3′ of NSE in ATM intron 28 of thepre-mRNA transcript or U2AF65 binding site upstream of the pseudoexon.

According to another aspect of the invention, there is provided a methodof tailoring functional ATM expression in a subject, cell or tissue,comprising the administration of a NSE-activator agent and/or aNSE-repressor agent described herein.

According to another aspect of the invention, there is provided use ofrs609261 genotyping to predict a subject response to therapy forconditions associated with ATM deregulation.

According to another aspect of the invention, there is provided acomposition comprising the NSE repressor agent of the invention herein.

According to another aspect of the invention, there is provided acomposition comprising the NSE activator agent of the invention herein.

According to another aspect of the invention, there is provided a methodof treatment or prevention of functional-ATM protein deficiency in asubject, the method comprising identifying the presence of a non-thyminevariant residue rs609261 located at position −3 relative to the 3′splice site of NSE (cryptic exon in ATM intron 28) of the human genome,wherein the presence of a non-thymine variant residue rs609261 indicatesthat the subject has, or is susceptible to, functional-ATM proteindeficiency, and administration of an agent to the subject, which isarranged to replace the non-thymine variant residue rs609261 with athymine residue.

According to another aspect of the invention, there is provided a methodof treatment or prevention of functional-ATM protein deficiency in asubject, the method comprising replacing a non-thymine variant residuers609261 located at position -3 relative to the 3′ splice site of NSE(cryptic exon in ATM intron 28) of the human genome with a thymineresidue.

According to another aspect of the invention, there is provided a vectorcomprising the polynucleic acid polymer of the invention.

According to another aspect of the invention, there is provided a methodof treatment or prevention of functional-ATM protein deficiency in asubject, the method comprising identifying the presence of a guaninevariant residue at rs4988000 of the human genome, wherein the presenceof a guanine variant residue at rs4988000 indicates that the subjecthas, or is susceptible to, functional-ATM protein deficiency, andadministration of an agent to the subject, which is arranged to replacethe guanine variant residue at rs4988000 with adenine.

According to another aspect of the invention, there is provided a methodof treatment or prevention of functional-ATM protein deficiency in asubject, the method comprising replacing a guanine variant residue atrs4988000 of the human genome with an adenine residue.

According to a first aspect of the invention, there is provided a methodof screening a subject or a population of subjects for susceptibility tofunctional-ATM protein deficiency, wherein the screening comprisesdetermining the presence of a guanine variant residue at rs4988000 ofthe human genome, wherein the presence of a guanine variant residue atrs4988000 indicates that the subject(or group of subjects) has, or issusceptible to, functional-ATM protein deficiency.

According to another aspect of the invention, there is provided a methodof selecting a subject or a population of subjects for treatment orprophylaxis, wherein the subject is susceptible to functional-ATMprotein deficiency, the method comprising determining the presence of aguanine variant residue at rs4988000 of the human genome, wherein thepresence of a guanine variant residue at rs4988000 indicates that thesubject has, or is susceptible to, functional-ATM protein deficiency,and selecting such subject for treatment with an agent arranged toincrease functional-ATM levels in the subject.

According to another aspect of the invention, there is provided a methodof treatment or prevention of functional-ATM protein deficiency in asubject, the method comprising identifying the presence of a guaninevariant residue at rs4988000 of the human genome, wherein the presenceof a guanine variant residue at rs4988000 indicates that the subjecthas, or is susceptible to, functional-ATM protein deficiency, andadministration of an agent to the subject, which is arranged to increasefunctional-ATM levels.

According to another aspect of the invention, there is provided a methodof screening for an agent or a combination of agents capable ofmodifying regulation of a gene's expression (FIG. 7) comprisingidentifying a nonsense-mediated RNA decay switch exon (NSE) that limitsfunctional gene expression; identifying one or more splicing regulatorymotifs upstream or downstream of the NSE that compete with the NSE forspliceosomal components, said regulatory motifs comprising crypticsplice sites or pseudo-exons; targeting the one or more splicingregulatory motifs with antisense polynucleic acid that are arranged tohybridize to the splicing regulatory motifs through Watson-Crick basepairing; and determining if there is an increased or decreased inclusionof the NSE in a mature RNA transcript of the gene.

According to another aspect of the invention, there is provided a methodof modulating gene's expression comprising providing an agent arrangedto bind to NSE splicing regulatory motifs.

According to another aspect of the invention, there is provided an agentarranged to bind to a gene splicing regulatory motif of NSE, wherein thesplicing regulatory motif controls inclusion of the NSE into a matureRNA transcript of the gene.

According to another aspect of the invention, provided herein is amethod of a treatment or prevention of a disease pathology caused by anNSE inclusion in an mRNA gene transcript comprising providing an agentarranged to bind to a gene NSE splicing regulatory motif that controlsinclusion of the NSE into a mature RNA transcript of the gene.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

The determination may use any suitable assay or genetic analysisavailable to the skilled person. In some instances, detection is done ata nucleic acid level with nucleic acid-based techniques such as in situhybridization and RT-PCR Sequencing technologies can includenext-generation sequencing technologies such as Helicos True SingleMolecule Sequencing (tSMS) (Harris T. D. et al., (2008) Science320:106-109); 454 sequencing (Roche) (Margulies, M. et al., 2005,Nature, 437, 376-380); SOLiD technology (Applied Biosystems); SOLEXAsequencing (Illumina); single molecule, real-time (SMRT™) technology ofPacific Biosciences; nanopore sequencing (Soni GV and Meller A. (2007)Clin Chem 53: 1996-2001); semiconductor sequencing (Ion Torrent;Personal Genome Machine); DNA nanoball sequencing; sequencing usingtechnology from Dover Systems (Polonator), and technologies that do notrequire amplification or otherwise transform native DNA prior tosequencing (e.g., Pacific Biosciences and Helicos), such asnanopore-based strategies (e.g., Oxford Nanopore, Genia Technologies,and Nabsys). Sequencing technologies can also include Sanger sequencing,Maxam-Gilbert sequencing, Shotgun sequencing, bridge PCR, massspectrometry based sequencing, microfluidic based Sanger sequencing,microscopy-based sequencing, RNAP sequencing, or hybridization basedsequencing.

Sequencing of a gene transcript of interest may also include anamplification step. Exemplary amplification methodologies include, butare not limited to, polymerase chain reaction (PCR), nucleic acidsequence based amplification (NASBA), self-sustained sequencereplication (3 SR), loop mediated isothermal amplification (LAMP),strand displacement amplification (SDA), whole genome amplification,multiple displacement amplification, strand displacement amplification,helicase dependent amplification, nicking enzyme amplification reaction,recombinant polymerase amplification, reverse transcription PCR,ligation mediated PCR, or methylation specific PCR.

Additional methods that can be used to obtain a nucleic acid sequenceinclude, e.g., whole-genome RNA expression array, enzyme-linkedimmunosorbent assay (ELISA), genome sequencing, de novo sequencing,Pacific Biosciences SMRT sequencing, immunohistochemistry (IHC),immunocytochemistry (ICC), mass spectrometry, tandem mass spectrometry,matrix-assisted laser desorption ionization time of flight massspectrometry (MALDI-TOF MS), in-situ hybridization, fluorescent in-situhybridization (FISH), chromogenic in-situ hybridization (CISH), silverin situ hybridization (SISH), digital PCR (dPCR), reverse transcriptionPCR, quantitative PCR (Q-PCR), single marker qPCR, real-time PCR,nCounter Analysis (Nanostring technology), Western blotting, Southernblotting, SDS-PAGE, gel electrophoresis, and Northern blotting.

According to another aspect of the invention, there is provided a methodof treatment or prevention of functional-ATM protein deficiency in asubject, the method comprising identifying the presence of a non-thyminevariant residue rs609261 located at position −3 relative to the 3′splice site of NSE (cryptic exon in ATM intron 28) of the human genome,wherein the presence of a non-thymine variant residue rs609261 indicatesthat the subject has, or is susceptible to, functional-ATM proteindeficiency, and administration of an agent to the subject, which isarranged to increase functional-ATM levels.

According to another aspect of the invention, there is provided a methodof treatment or prevention of a condition associated with afunctional-ATM protein deficiency, comprising the administration of aNSE repressor agent arranged to increase levels of functional ATMprotein, wherein the agent is arranged to bind to a NSE in ATM intron 28of the pre-mRNA transcript to decrease inclusion of the NSE in themature RNA transcript.

Decreasing inclusion of the NSE in the mature RNA transcript may providean increase in functional ATM protein expression.

The method of treatment or prevention of functional-ATM proteindeficiency in a subject or an at-risk population of subjects may be amethod of treatment or prevention of a condition associated withfunctional-ATM protein deficiency. The condition may be any symptom ofataxia-telangiectasia; cerebellar ataxia; oculocutaneous angiectasia;cancer; immune deficiency; cellular radiosensitivity; or chromosomalinstability. The cancer may comprise lymphoblastoid leukemias, orlymphomas. In one embodiment, the condition is ataxia-telangiectasia. Inanother embodiment, the condition is cancer. The cancer may comprise anon-Hodgkin or Hodgkin lymphoma.

In one embodiment, the NSE comprises the sequencetctacaggttggctgcatagaagaaaaag (SEQ ID NO: 57). The NSE repressor agentmay be arranged to bind to NSE within the sequenceagTCTACAGGTTGGCTGCATAGAAGAAAAAGgtagag (SEQ ID NO: 58) (respective 3′ and5′ splice site dinucleotides of flanking intervening sequences areunderlined). The NSE repressor agent may be arranged to bind to the 5′or 3′ splice site of the NSE in ATM intron 28. In another embodiment,the NSE repressor agent is arranged to bind to the 3′ splice site of theNSE in ATM intron 28. In another embodiment, the NSE repressor agent maybe arranged to bind to NSE within the sequencetcttagTCTACAGGTTGGCTGCATAGAAGAAAAAGgtagag (SEQ ID NO: 59) (respective 3′and 5′ splice site dinucleotides of flanking intervening sequences areunderlined). In another embodiment, the NSE repressor agent may bearranged to bind to NSE within the sequencetctcagTCTACAGGTTGGCTGCATAGAAGAAAAAGgtagag (SEQ ID NO: 60) (respective 3′and 5′ splice site dinucleotides of flanking intervening sequences areunderlined).

According to another aspect of the invention, there is provided a methodof treatment or prevention of a condition associated with deregulationof ATM expression in a subject comprising the administration of aNSE-activator agent, wherein the NSE-activator agent is arranged toincrease NSE inclusion in ATM mature RNA transcript by binding tosplicing regulatory motifs in ATM intron 28.

Increasing inclusion of the NSE in the mature RNA transcript may providea decrease in functional ATM protein expression.

According to another aspect of the invention, there is provided a methodof treatment or prevention of cancer in a subject comprising theadministration of a NSE-activator agent arranged to increase a cancercell's susceptibility to cytotoxic therapy with DNA damaging agents suchas radiotherapy, wherein the NSE-activator agent is arranged to increaseNSE inclusion in ATM mature RNA transcript by binding to splicingregulatory motifs in ATM intron 28; and treating the subject with thecytotoxic therapy, such as radiotherapy or chemotherapy.

Chemotherapy may comprise a therapeutic that induces double strand DNAbreaks. The skilled person will understand that there are severalchemotherapy/therapeutic agents that are capable of inducing doublestrand DNA breaks. In one embodiment, the chemotherapy agents maycomprise bleomycin.

Increasing inclusion of the NSE in the mature RNA transcript may providea decrease in functional ATM protein expression.

The radiotherapy or chemotherapy may be following the administration ofthe agent The radiotherapy or chemotherapy may one or more daysfollowing the administration of the agent The radiotherapy orchemotherapy may be one or more weeks following the administration ofthe agent

In one embodiment the pseudoexon comprises the sequencetcatcgaatacttttggaaataag (SEQ ID NO: 61).

According to another aspect of the invention, there is provided a methodof increasing a cell's susceptibility to cytotoxic therapy with DNAdamaging agents such as radiotherapy comprising the reduction of ATMprotein expression by administration of a NSE-activator agent arrangedto increase NSE inclusion in ATM mature RNA transcript by binding to NSEregulatory motifs in ATM intron 28.

In one embodiment the cell is a cancerous cell. In another embodimentthe cell is a pre-cancerous cell.

According to another aspect of the invention, there is provided a methodof tailoring functional ATM expression in a subject, cell or tissue,comprising the administration of a NSE-activator agent and/or aNSE-repressor agent described herein.

Nonsense-Mediated mRNA Decay

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that existsin all eukaryotes. Its main function is to reduce errors in geneexpression by eliminating mRNA transcripts that contain premature stopcodons. NMD targets transcripts with premature stop codons but also abroad array of mRNA isoforms expressed from many endogenous genes,suggesting that NMD is a master regulator that drives both fine andcoarse adjustments in steady-state RNA levels in the cell.

A nonsense-mediated RNA decay switch exon (NSE) is an exon or apseudoexon that activates the NMD pathway if included in a mature RNAtranscript A NSE inclusion in mature transcripts downregulates geneexpression.

Cryptic (or pseudo-splice sites) have the same splicing recognitionsequences as genuine splice sites but are not used in the splicingreactions. They outnumber genuine splice sites in the human genome by anorder of a magnitude and are normally repressed by thus far poorlyunderstood molecular mechanisms. Cryptic 5′ splice sites have theconsensus NNN/GUNNNN or NNN/GCNNNN where N is any nucleotide and / isthe exon-intron boundary. Cryptic 3′ splice sites have the consensusNAG/N. Their activation is positively influenced by surroundingnucleotides that make them more similar to the optimal consensus ofauthentic splice sites, namely MAG/GURAGU and YAG/G, respectively, whereM is C or A, R is G or A, and Y is C or U.

Cryptic (or pseudo-) exons have the same splicing recognition sequencesas genuine exons but are not used in the splicing reactions. Theyoutnumber genuine exons by an order of a magnitude and are normallyrepressed by thus far poorly understood molecular mechanisms.

Splice sites and their regulatory sequences can be readily identified bya skilled person using suitable algorithms publicly available, listedfor example in Kralovicova, J. and Vorechovsky, I. (2007) Global controlof aberrant splice site activation by auxiliary splicing sequences:evidence for a gradient in exon and intron definition. Nucleic AcidsRes., 35, 6399-6413,(http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2095810/pdf/gkm680.pdf).

The cryptic splice sites or splicing regulatory sequences may competefor RNA-binding proteins such as U2AF with a splice site of the NSE. Inone embodiment, the agent may bind to the cryptic splice site orsplicing regulatory sequences to prevent the binding of RNA-bindingproteins and thereby favoring utilization of the NSE splice sites.

In one embodiment, the cryptic splice site may not comprise the 5′ or 3′splice site of the NSE. The cryptic splice site may be at least 10nucleotides upstream of the NSE 5′ splice site. The cryptic splice sitemay be at least 20 nucleotides upstream of the NSE 5′ splice site. Thecryptic splice site may be at least 50 nucleotides upstream of the NSE5′ splice site. The cryptic splice site may be at least 100 nucleotidesupstream of the NSE 5′ splice site. The cryptic splice site may be atleast 200 nucleotides upstream of the NSE 5′ splice site.

The cryptic splice site may be at least 10 nucleotides downstream of theNSE 3′ splice site. The cryptic splice site may be at least 20nucleotides downstream of the NSE 3′ splice site. The cryptic splicesite may be at least 50 nucleotides downstream of the NSE 3′ splicesite. The cryptic splice site may be at least 100 nucleotides downstreamof the NSE 3′ splice site. The cryptic splice site may be at least 200nucleotides downstream of the NSE 3′ splice site.

The NSE Repressor Agent and NSE Activator Agent

The NSE repressor agent and/or NSE activator agent may comprise apolynucleic acid polymer. In one embodiment, the NSE repressor agentand/or NSE activator agent is an SSO (Splice Switching Oligonucleotide).

In an embodiment wherein the NSE repressor agent and/or NSE activatoragent comprises a polynucleic acid polymer the following statements mayapply equally to both the NSE repressor agent and the NSE activatoragent unless otherwise indicated. The polynucleic acid polymer may beabout 50 nucleotides in length. The polynucleic acid polymer may beabout 45 nucleotides in length. The polynucleic acid polymer may beabout 40 nucleotides in length. The polynucleic acid polymer may beabout 35 nucleotides in length. The polynucleic acid polymer may beabout 30 nucleotides in length. The polynucleic acid polymer may beabout 24 nucleotides in length. The polynucleic acid polymer may beabout 25 nucleotides in length. The polynucleic acid polymer may beabout 20 nucleotides in length. The polynucleic acid polymer may beabout 19 nucleotides in length. The polynucleic acid polymer may beabout 18 nucleotides in length. The polynucleic acid polymer may beabout 17 nucleotides in length. The polynucleic acid polymer may beabout 16 nucleotides in length. The polynucleic acid polymer may beabout 15 nucleotides in length. The polynucleic acid polymer may beabout 14 nucleotides in length. The polynucleic acid polymer may beabout 13 nucleotides in length. The polynucleic acid polymer may beabout 12 nucleotides in length. The polynucleic acid polymer may beabout 11 nucleotides in length. The polynucleic acid polymer may beabout 10 nucleotides in length. The polynucleic acid polymer may bebetween about 10 and about 50 nucleotides in length. The polynucleicacid polymer may be between about 10 and about 45 nucleotides in length.The polynucleic acid polymer may be between about 10 and about 40nucleotides in length. The polynucleic acid polymer may be between about10 and about 35 nucleotides in length. The polynucleic acid polymer maybe between about 10 and about 30 nucleotides in length. The polynucleicacid polymer may be between about 10 and about 25 nucleotides in length.The polynucleic acid polymer may be between about 10 and about 20nucleotides in length. The polynucleic acid polymer may be between about15 and about 25 nucleotides in length. The polynucleic acid polymer maybe between about 15 and about 30 nucleotides in length. The polynucleicacid polymer may be between about 12 and about 30 nucleotides in length.

The sequence of the polynucleic acid polymer may be at least 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 99.5% complementary to a target sequence of the partiallyprocessed mRNA transcript. The sequence of the polynucleic acid polymermay be 100% complementary to a target sequence of the pre-mRNAtranscript.

The sequence of the polynucleic acid polymer may have 4 or lessmismatches to a target sequence of the pre-mRNA transcript. The sequenceof the polynucleic acid polymer may have 3 or less mismatches to atarget sequence of the pre-mRNA transcript. The sequence of thepolynucleic acid polymer may have 2 or less mismatches to a targetsequence of the pre-mRNA transcript. The sequence of the polynucleicacid polymer may have 1 or less mismatches to a target sequence of thepre-mRNA transcript.

The polynucleic acid polymer may specifically hybridize to a targetsequence of the pre-mRNA transcript. The specificity may be at least a91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequencecomplementarity of the polynucleic acid polymer to a target sequence ofthe pre-mRNA transcript The hybridization may be under high stringenthybridization conditions.

The polynucleic acid polymer may have a sequence with at least 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 99.5% sequence identity to a sequence illustrated in Table2 or FIG. 20. The polynucleic acid polymer may have a sequence with 100%sequence identity to a sequence illustrated in Table 2 or FIG. 20. Insome instances, the polynucleic acid polymer may have a sequence with atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to a sequenceillustrated in Table 2. In some cases, the polynucleic acid polymer mayhave a sequence with 100% sequence identity to a sequence illustrated inTable 2.

In some instances, the polynucleic acid polymer has a sequence with atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to a sequenceselected from SEQ ID NOs: 18-52. In some cases, the polynucleic acidpolymer has a sequence with at least 50% sequence identity to a sequenceselected from SEQ ID NOs: 18-52. In some cases, the polynucleic acidpolymer has a sequence with at least 60% sequence identity to a sequenceselected from SEQ ID NOs: 18-52. In some cases, the polynucleic acidpolymer has a sequence with at least 70% sequence identity to a sequenceselected from SEQ ID NOs: 18-52. In some cases, the polynucleic acidpolymer has a sequence with at least 80% sequence identity to a sequenceselected from SEQ ID NOs: 18-52. In some cases, the polynucleic acidpolymer has a sequence with at least 85% sequence identity to a sequenceselected from SEQ ID NOs: 18-52. In some cases, the polynucleic acidpolymer has a sequence with at least 90% sequence identity to a sequenceselected from SEQ ID NOs: 18-52. In some cases, the polynucleic acidpolymer has a sequence with at least 91% sequence identity to a sequenceselected from SEQ ID NOs: 18-52. In some cases, the polynucleic acidpolymer has a sequence with at least 92% sequence identity to a sequenceselected from SEQ ID NOs: 18-52. In some cases, the polynucleic acidpolymer has a sequence with at least 93% sequence identity to a sequenceselected from SEQ ID NOs: 18-52. In some cases, the polynucleic acidpolymer has a sequence with at least 94% sequence identity to a sequenceselected from SEQ ID NOs: 18-52. In some cases, the polynucleic acidpolymer has a sequence with at least 95% sequence identity to a sequenceselected from SEQ ID NOs: 18-52. In some cases, the polynucleic acidpolymer has a sequence with at least 96% sequence identity to a sequenceselected from SEQ ID NOs: 18-52. In some cases, the polynucleic acidpolymer has a sequence with at least 97% sequence identity to a sequenceselected from SEQ ID NOs: 18-52. In some cases, the polynucleic acidpolymer has a sequence with at least 98% sequence identity to a sequenceselected from SEQ ID NOs: 18-52. In some cases, the polynucleic acidpolymer has a sequence with at least 99% sequence identity to a sequenceselected from SEQ ID NOs: 18-52. In some cases, the polynucleic acidpolymer has a sequence with at least 99.5% sequence identity to asequence selected from SEQ ID NOs: 18-52. In some cases, the polynucleicacid polymer has a sequence with 100% sequence identity to a sequenceselected from SEQ ID NOs: 18-52.

In some embodiments, a polynucleic acid polymer hybridizes to a motifwithin a transposed element, upstream of a transposed element, ordownstream of a transposed element. In some instances, the transposedelement is Alu, MER51, UC or L4C. In some instances, the transposedelement is Alu (e.g., Alu− or Alu+) or MER51. In some cases, thetransposed element is Alu (e.g., Alu− or Alu+). In other cases, thetransposed element is MER51. In some instances, the polynucleic acidpolymer hybridizes to a target motif within Alu (e.g., Alu− or Alu+). Inother instances, the polynucleic acid polymer hybridizes to a targetmotif downstream of MER51. In some instances, the polynucleic acidpolymer has a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequenceidentity to a sequence selected from SEQ ID NOs: 18-52.

In some embodiments, the polynucleic acid polymer hybridizes to a targetmotif that is either upstream or downstream of Alu (e.g., Alu− or Alu+).In some instances, the polynucleic acid polymer hybridizes to a targetmotif that is upstream of Alu. In some cases, the target motif is about5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110,120, 130, 140, 150, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700,800, or more bases upstream of Alu. In some cases, the target motif isabout 5 or more bases upstream of Alu. In some cases, the target motifis about 10 or more bases upstream of Alu. In some cases, the targetmotif is about 20 or more bases upstream of Alu. In some cases, thetarget motif is about 30 or more bases upstream of Alu. In some cases,the target motif is about 40 or more bases upstream of Alu. In somecases, the target motif is about 50 or more bases upstream of Alu. Insome cases, the target motif is about 80 or more bases upstream of Alu.In some cases, the target motif is about 100 or more bases upstream ofAlu. In some cases, the target motif is about 150 or more bases upstreamof Alu. In some cases, the target motif is about 200 or more basesupstream of Alu. In some cases, the target motif is about 300 or morebases upstream of Alu. In some cases, the target motif is about 500 ormore bases upstream of Alu. In some cases, the target motif is about 800or more bases upstream of Alu. In some instances, the polynucleic acidpolymer has a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequenceidentity to a sequence selected from SEQ ID NOs: 18-52.

In some instances, the polynucleic acid polymer hybridizes to a targetmotif that is downstream of Alu (e.g., Alu− or Alu+). In some cases, thetarget motif is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70,80, 90, 100, 110, 120, 130, 140, 150, 180, 200, 250, 300, 350, 400, 450,500, 600, 700, 800, or more bases downstream of Alu. In some cases, thetarget motif is about 5 or more bases downstream of Alu. In some cases,the target motif is about 10 or more bases downstream of Alu. In somecases, the target motif is about 20 or more bases downstream of Alu. Insome cases, the target motif is about 30 or more bases downstream ofAlu. In some cases, the target motif is about 40 or more basesdownstream of Alu. In some cases, the target motif is about 50 or morebases downstream of Alu. In some cases, the target motif is about 80 ormore bases downstream of Alu. In some cases, the target motif is about100 or more bases downstream of Alu. In some cases, the target motif isabout 150 or more bases downstream of Alu. In some cases, the targetmotif is about 200 or more bases downstream of Alu. In some cases, thetarget motif is about 300 or more bases downstream of Alu. In somecases, the target motif is about 500 or more bases downstream of Alu. Insome cases, the target motif is about 800 or more bases downstream ofAlu. In some instances, the polynucleic acid polymer has a sequence withat least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to a sequenceselected from SEQ ID NOs: 18-52.

In some embodiments, the polynucleic acid polymer hybridizes to a targetmotif that is either upstream or downstream of MER51. In some instances,the polynucleic acid polymer hybridizes to a target motif that isupstream of MER51. In some cases, the target motif is about 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 120, 130, 140,150, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, or morebases upstream of MER51. In some cases, the target motif is about 5 ormore bases upstream of MER51. In some cases, the target motif is about10 or more bases upstream of MER51. In some cases, the target motif isabout 20 or more bases upstream of MER51. In some cases, the targetmotif is about 30 or more bases upstream of MER51. In some cases, thetarget motif is about 40 or more bases upstream of MER51. In some cases,the target motif is about 50 or more bases upstream of MER51. In somecases, the target motif is about 80 or more bases upstream of MER51. Insome cases, the target motif is about 100 or more bases upstream ofMER51. In some cases, the target motif is about 150 or more basesupstream of MER51. In some cases, the target motif is about 200 or morebases upstream of MER51. In some cases, the target motif is about 300 ormore bases upstream of MER51. In some cases, the target motif is about500 or more bases upstream of MER51. In some cases, the target motif isabout 800 or more bases upstream of MER51. In some instances, thepolynucleic acid polymer has a sequence with at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 99.5% sequence identity to a sequence selected from SEQ ID NOs:18-52.

In some instances, the polynucleic acid polymer hybridizes to a targetmotif that is downstream of MER51. In some cases, the target motif isabout 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100,110, 120, 130, 140, 150, 180, 200, 250, 300, 350, 400, 450, 500, 600,700, 800, or more bases downstream of MER51. In some cases, the targetmotif is about 5 or more bases downstream of MER51. In some cases, thetarget motif is about 10 or more bases downstream of MER51. In somecases, the target motif is about 20 or more bases downstream of MER51.In some cases, the target motif is about 30 or more bases downstream ofMER51. In some cases, the target motif is about 40 or more basesdownstream of MER51. In some cases, the target motif is about 50 or morebases downstream of MER51. In some cases, the target motif is about 80or more bases downstream of MER51. In some cases, the target motif isabout 100 or more bases downstream of MER51. In some cases, the targetmotif is about 150 or more bases downstream of MER51. In some cases, thetarget motif is about 200 or more bases downstream of MER51. In somecases, the target motif is about 300 or more bases downstream of MER51.In some cases, the target motif is about 500 or more bases downstream ofMER51. In some cases, the target motif is about 800 or more basesdownstream of MER51. In some instances, the polynucleic acid polymer hasa sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identityto a sequence selected from SEQ ID NOs: 18-52.

Where reference is made to a polynucleic acid polymer sequence, theskilled person will understand that one or more substitutions may betolerated, optionally two substitutions may be tolerated in thesequence, such that it maintains the ability to hybridize to the targetsequence, or where the substitution is in a target sequence, the abilityto be recognized as the target sequence. References to sequence identitymay be determined by BLAST sequence alignment(www.ncbi.nlm.nih.gov/BLAST/) using standard/default parameters. Forexample, the sequence may have 99% identity and still function accordingto the invention. In other embodiments, the sequence may have 98%identity and still function according to the invention. In anotherembodiment, the sequence may have 95% identity and still functionaccording to the invention.

A polynucleic acid polymer, such as the SSOs, may comprise RNA or DNA.The polynucleic acid polymer, such as the SSOs, may comprise RNA. Thepolynucleic acid polymer, such as the SSOs, may comprise natural orsynthetic or artificial nucleotide analogues or bases, having equivalentcomplementation as DNA or RNA. The polynucleic acid polymer, such as theSSOs, may comprise combinations of DNA, RNA and/or nucleotide analogues.Nucleotide analogues may comprise PNA or LNA. In another embodiment, thenucleic acid, such as the SSOs, may comprise or consist of PMO.

In some instances, the synthetic or artificial nucleotide analogues orbases can comprise modifications at one or more of ribose moiety,phosphate moiety, nucleoside moiety, or a combination thereof Forexample, a nucleotide base may be any naturally occurring, unmodifiednucleotide base such as adenine, guanine, cytosine, thymine and uracil,or any synthetic or modified base that is sufficiently similar to anunmodified nucleotide base such that it is capable of hydrogen bondingwith a base present on a target pre-mRNA. Examples of modifiednucleotide bases include, without limitation, hypoxanthine, xanthine,7-methylguanine, 5,6-dihydrouracil, 5-methyl cytosine, and5-hydroxymethoylcytosine.

Sometimes, the polynucleic acid polymers described herein also comprisea backbone structure that connects the components of an oligomer. Theterm “backbone structure” and “oligomer linkages” may be usedinterchangeably and refer to the connection between monomers of thepolynucleic acid polymer. In naturally occurring oligonucleotides, thebackbone comprises a 3′-5′ phosphodiester linkage connecting sugarmoieties of the oligomer. The backbone structure or oligomer linkages ofthe polynucleic acid polymers described herein may include (but are notlimited to) phosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate,phosphoramidate, and the like. See e.g., LaPlanche et al., Nucleic AcidsRes. 14:9081 (1986); Stec et al. , J. Am. Chem. Soc. 106:6077 (1984),Stein et al. , Nucleic Acids Res. 16:3209 (1988), Zon et al. , AntiCancer Drug Design 6:539 (1991); Zon et al. , Oligonucleotides andAnalogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., OxfordUniversity Press, Oxford England (1991)); Stec et al., U.S. Pat. No.5,151,510; Uhlmann and Peyman, Chemical Reviews 90:543 (1990).

In embodiments, the stereochemistry at each of the phosphorusinternucleotide linkages of the polynucleic acid polymer backbone israndom. In embodiments, the stereochemistry at each of the phosphorusinternucleotide linkages of the polynucleic acid polymer backbone iscontrolled and is not random. For example, U.S. Pat. App. Pub. No.2014/0194610, “Methods for the Synthesis of Functionalized NucleicAcids,” incorporated herein by reference, describes methods forindependently selecting the handedness of chirality at each phosphorousatom in a nucleic acid oligomer. In embodiments, a polynucleic acidpolymer described herein comprises a polynucleic acid polymer havingphosphorus internucleotide linkages that are not random. In embodiments,a composition used in the methods of the invention comprises a purediastereomeric polynucleic acid polymer. In embodiments, a compositionused in the methods of the invention comprises a polynucleic acidpolymer that has diastereomeric purity of at least about 90%, at leastabout 91%, at least about 92%, at least about 93%, at least about 94%,at least about 95%, at least about 96%, at least about 97%, at leastabout 98%, at least about 99%, about 100%, about 90% to about 100%,about 91% to about 100%, about 92% to about 100%, about 93% to about100%, about 94% to about 100%, about 95% to about 100%, about 96% toabout 100%, about 97% to about 100%, about 98% to about 100%, or about99% to about 100%.

In embodiments, the polynucleic acid polymer has a nonrandom mixture ofRp and Sp configurations at its phosphorus internucleotide linkages. Forexample, a mix of Rp and Sp may be required in antisenseoligonucleotides to achieve a balance between good activity and nucleasestability (Wan, et al. , 2014, “Synthesis, biophysical properties andbiological activity of second generation antisense oligonucleotidescontaining chiral phosphorothioate linkages,” Nucleic Acids Research42(22): 13456-13468, incorporated herein by reference). In embodiments,a polynucleic acid polymer described herein comprises about 5-100% Rp,at least about 5% Rp, at least about 10% Rp, at least about 15% Rp, atleast about 20% Rp, at least about 25% Rp, at least about 30% Rp, atleast about 35% Rp, at least about 40% Rp, at least about 45% Rp, atleast about 50% Rp, at least about 55% Rp, at least about 60% Rp, atleast about 65% Rp, at least about 70% Rp, at least about 75% Rp, atleast about 80% Rp, at least about 85% Rp, at least about 90% Rp, or atleast about 95% Rp, with the remainder Sp, or about 100% Rp. Inembodiments, a polynucleic acid polymer described herein comprises about10% to about 100% Rp, about 15% to about 100% Rp, about 20% to about100% Rp, about 25% to about 100% Rp, about 30% to about 100% Rp, about35% to about 100% Rp, about 40% to about 100% Rp, about 45% to about100% Rp, about 50% to about 100% Rp, about 55% to about 100% Rp, about60% to about 100% Rp, about 65% to about 100% Rp, about 70% to about100% Rp, about 75% to about 100% Rp, about 80% to about 100% Rp, about85% to about 100% Rp, about 90% to about 100% Rp, or about 95% to about100% Rp, about 20% to about 80% Rp, about 25% to about 75% Rp, about 30%to about 70% Rp, about 40% to about 60% Rp, or about 45% to about 55%Rp, with the remainder Sp.

In embodiments, a polynucleic acid polymer described herein comprisesabout 5-100% Sp, at least about 5% Sp, at least about 10% Sp, at leastabout 15% Sp, at least about 20% Sp, at least about 25% Sp, at leastabout 30% Sp, at least about 35% Sp, at least about 40% Sp, at leastabout 45% Sp, at least about 50% Sp, at least about 55% Sp, at leastabout 60% Sp, at least about 65% Sp, at least about 70% Sp, at leastabout 75% Sp, at least about 80% Sp, at least about 85% Sp, at leastabout 90% Sp, or at least about 95% Sp, with the remainder Rp, or about100% Sp. In embodiments, a polynucleic acid polymer described hereincomprises about 10% to about 100% Sp, about 15% to about 100% Sp, about20% to about 100% Sp, about 25% to about 100% Sp, about 30% to about100% Sp, about 35% to about 100% Sp, about 40% to about 100% Sp, about45% to about 100% Sp, about 50% to about 100% Sp, about 55% to about100% Sp, about 60% to about 100% Sp, about 65% to about 100% Sp, about70% to about 100% Sp, about 75% to about 100% Sp, about 80% to about100% Sp, about 85% to about 100% Sp, about 90% to about 100% Sp, orabout 95% to about 100% Sp, about 20% to about 80% Sp, about 25% toabout 75% Sp, about 30% to about 70% Sp, about 40% to about 60% Sp, orabout 45% to about 55% Sp, with the remainder Rp.

Nucleotide analogues or artificial nucleotide base may comprise anucleic acid with a modification at a 2′ hydroxyl group of the ribosemoiety. The modification can be a 2′-O-methyl modification or a2′-O-methoxyethyl (2′-O-MOE) modification. The 2′-O-methyl modificationcan add a methyl group to the 2′ hydroxyl group of the ribose moietywhereas the 2′O-methoxyethyl modification can add a methoxyethyl groupto the 2′ hydroxyl group of the ribose moiety. Exemplary chemicalstructures of a 2′-O-methyl modification of an adenosine molecule and2′O-methoxyethyl modification of an uridine are illustrated below.

An additional modification at the 2′ hydroxyl group can include a2′-O-aminopropyl sugar conformation which can involve an extended aminegroup comprising a propyl linker that binds the amine group to the 2′oxygen. This modification can neutralize the phosphate derived overallnegative charge of the oligonucleotide molecule by introducing onepositive charge from the amine group per sugar and can thereby improvecellular uptake properties due to its zwitterionic properties. Anexemplary chemical structure of a 2′-O-aminopropyl nucleosidephosphoramidite is illustrated below.

Another modification at the 2′ hydroxyl group can include a locked orbridged ribose conformation (e.g., locked nucleic acid or LNA) where the4′ ribose position can also be involved. In this modification, theoxygen molecule bound at the 2′ carbon can be linked to the 4′ carbon bya methylene group, thus forming a 2′-C,4′-C-oxy-methylene-linkedbicyclic ribonucleotide monomer. Exemplary representations of thechemical structure of LNA are illustrated below. The representationshown to the left highlights the chemical connections of an LNA monomer.The representation shown to the right highlights the locked 3′-endo (3E)conformation of the furanose ring of an LNA monomer.

A further modification at the 2′ hydroxyl group may comprise ethylenenucleic acids (ENA) such as for example 2′-4′-ethylene-bridged nucleicacid, which locks the sugar conformation into a C3′-endo sugar puckeringconformation. ENA are part of the bridged nucleic acids class ofmodified nucleic acids that also comprises LNA. Exemplary chemicalstructures of the ENA and bridged nucleic acids are illustrated below.

Still other modifications at the 2′ hydroxyl group can include 2′-deoxy,T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O-N-methylacetamido(2′-O-NMA).

Nucleotide analogues may further comprise Morpholinos, peptide nucleicacids (PNAs), methylphosphonate nucleotides, thiolphosphonatenucleotides, 2′-fluoro N3-P5′-phosphoramidites, 1′,5′-anhydrohexitolnucleic acids (HNAs), or a combination thereof. Morpholino orphosphorodiamidate morpholino oligo (PMO) comprises synthetic moleculeswhose structure mimics natural nucleic acid structure by deviates fromthe normal sugar and phosphate structures. Instead, the five memberribose ring can be substituted with a six member morpholino ringcontaining four carbons, one nitrogen and one oxygen. The ribosemonomers can be linked by a phosphordiamidate group instead of aphosphate group. These backbone alterations can remove all positive andnegative charges making morpholinos neutral molecules that can crosscellular membranes without the aid of cellular delivery agents such asthose used by charged oligonucleotides.

Peptide nucleic acid (PNA) does not contain sugar ring or phosphatelinkage. Instead, the bases can be attached and appropriately spaced byoligoglycine-like molecules, therefore, eliminating a backbone charge.

Modification of the phosphate backbone may also comprise methyl or thiolmodifications such as methylphosphonate nucleotide and. Exemplarythiolphosphonate nucleotide (left) and methylphosphonate nucleotide(right) are illustrated below.

Furthermore, exemplary 2′-fluoro N3-P5′-phosphoramidites is illustratedas:

And exemplary hexitol nucleic acid (or 1′,5′-anhydrohexitol nucleicacids (HNA)) is illustrated as:

In addition to modification of the ribose moiety, phosphate backbone andthe nucleoside, the nucleotide analogues can also be modified by forexample at the 3′ or the 5′ terminus. For example, the 3′ terminus caninclude a 3′ cationic group, or by inverting the nucleoside at the3′-terminus with a 3′-3′ linkage. In another alternative, the3′-terminus can be blocked with an aminoalkyl group, e.g., a 3′C5-aminoalkyl dT. The 5′-terminus can be blocked with an aminoalkylgroup, e.g., a 5′-O-alkylamino substituent. Other 5′ conjugates caninhibit 5′-3′ exonucleolytic cleavage. Other 3′ conjugates can inhibit3′-5′ exonucleolytic cleavage.

Unless specified otherwise, the left-hand end of single-stranded nucleicacid (e.g., pre-mRNA transcript, oligonucleotide, SSO, etc.) sequencesis the 5′ end and the left-hand direction of single or double-strandednucleic acid sequences is referred to as the 5′ direction. Similarly,the right-hand end or direction of a nucleic acid sequence (single ordouble stranded) is the 3′ end or direction. Generally, a region orsequence that is 5′ to a reference point in a nucleic acid is referredto as “upstream,” and a region or sequence that is 3′ to a referencepoint in a nucleic acid is referred to as “downstream.” Generally, the5′ direction or end of an mRNA is where the initiation or start codon islocated, while the 3′ end or direction is where the termination codon islocated. In some aspects, nucleotides that are upstream of a referencepoint in a nucleic acid may be designated by a negative number, whilenucleotides that are downstream of a reference point may be designatedby a positive number. For example, a reference point (e.g., an exon-exonjunction in mRNA) may be designated as the “zero” site, and a nucleotidethat is directly adjacent and upstream of the reference point isdesignated “minus one,” e.g., “−1,” while a nucleotide that is directlyadjacent and downstream of the reference point is designated “plus one,”e.g., “+1.”

In some cases, one or more of the artificial nucleotide analoguesdescribed herein are resistant toward nucleases such as for exampleribonuclease such as RNase H, deoxyribonuclease such as DNase, orexonuclease such as 5′-3′ exonuclease and 3′-5′ exonuclease whencompared to natural polynucleic acid polymers. In some instances,artificial nucleotide analogues comprising 2′-O-methyl,2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy,T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O-N-methylacetamido(2′-O-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonatenucleotides, thiolphosphonate nucleotides, 2′-fluoroN3-P5′-phosphoramidites, or combinations thereof are resistant towardnucleases such as for example ribonuclease such as RNase H,deoxyribonuclease such as DNase, or exonuclease such as 5′-3′exonuclease and 3′-5′ exonuclease. 2′-O-methyl modified polynucleic acidpolymer may be nuclease resistance (e.g., RNase H, DNase, 5′-3′exonuclease or 3′-5′ exonuclease resistance). 2′O-methoxyethyl(2′-O-MOE) modified polynucleic acid polymer may be nuclease resistance(e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonucleaseresistance). 2′-O-aminopropyl modified polynucleic acid polymer may benuclease resistance (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′exonuclease resistance). 2′-deoxy modified polynucleic acid polymer maybe nuclease resistance (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′exonuclease resistance). T-deoxy-2′-fluoro modified polynucleic acidpolymer may be nuclease resistance (e.g., RNase H, DNase, 5′-3′exonuclease or 3′-5′ exonuclease resistance). 2′-O-aminopropyl (2′-O-AP)modified polynucleic acid polymer may be nuclease resistance (e.g.,RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistance).2′-O-dimethylaminoethyl (2′-O-DMAOE) modified polynucleic acid polymermay be nuclease resistance (e.g., RNase H, DNase, 5′-3′ exonuclease or3′-5′ exonuclease resistance). 2′-O-dimethylaminopropyl (2′-O-DMAP)modified polynucleic acid polymer may be nuclease resistance (e.g.,RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistance).T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE) modified polynucleic acidpolymer may be nuclease resistance (e.g., RNase H, DNase, 5′-3′exonuclease or 3′-5′ exonuclease resistance). 2′-O—N-methylacetamido(2′-O-NMA) modified polynucleic acid polymer may be nuclease resistance(e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonucleaseresistance). LNA modified polynucleic acid polymer may be nucleaseresistance (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonucleaseresistance). ENA modified polynucleic acid polymer may be nucleaseresistance (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonucleaseresistance). HNA modified polynucleic acid polymer may be nucleaseresistance (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonucleaseresistance). Morpholinos may be nuclease resistance (e.g., RNase H,DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistance). PNA can beresistant to nucleases (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′exonuclease resistance). Methylphosphonate nucleotides modifiedpolynucleic acid polymer may be nuclease resistance (e.g., RNase H,DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistance).Thiolphosphonate nucleotides modified polynucleic acid polymer may benuclease resistance (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′exonuclease resistance). Polynucleic acid polymer comprising 2′-fluoroN3-P5′-phosphoramidites may be nuclease resistance (e.g., RNase H,DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistance).

In some instances, one or more of the artificial nucleotide analoguesdescribed herein have increased binding affinity toward their mRNAtarget relative to an equivalent natural polynucleic acid polymer. Theone or more of the artificial nucleotide analogues comprising2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy,T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O-N-methylacetamido(2′-O-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonatenucleotides, thiolphosphonate nucleotides, or 2′-fluoroN3-P5′-phosphoramidites can have increased binding affinity toward theirmRNA target relative to an equivalent natural polynucleic acid polymer.2′-O-methyl modified polynucleic acid polymer can have increased bindingaffinity toward their mRNA target relative to an equivalent naturalpolynucleic acid polymer. 2′-O-methoxyethyl (2′-O-MOE) modifiedpolynucleic acid polymer can have increased binding affinity towardtheir mRNA target relative to an equivalent natural polynucleic acidpolymer. 2′-O-aminopropyl modified polynucleic acid polymer can haveincreased binding affinity toward their mRNA target relative to anequivalent natural polynucleic acid polymer. 2′-deoxy modifiedpolynucleic acid polymer can have increased binding affinity towardtheir mRNA target relative to an equivalent natural polynucleic acidpolymer. T-deoxy-2′-fluoro modified polynucleic acid polymer can haveincreased binding affinity toward their mRNA target relative to anequivalent natural polynucleic acid polymer. 2′-O-aminopropyl (2′-O-AP)modified polynucleic acid polymer can have increased binding affinitytoward their mRNA target relative to an equivalent natural polynucleicacid polymer. 2′-O-dimethylaminoethyl (2′-O-DMAOE) modified polynucleicacid polymer can have increased binding affinity toward their mRNAtarget relative to an equivalent natural polynucleic acid polymer.2′-O-dimethylaminopropyl (2′-O-DMAP) modified polynucleic acid polymercan have increased binding affinity toward their mRNA target relative toan equivalent natural polynucleic acid polymer.T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE) modified polynucleic acidpolymer can have increased binding affinity toward their mRNA targetrelative to an equivalent natural polynucleic acid polymer.2′-O-N-methylacetamido (2′-O-NMA) modified polynucleic acid polymer canhave increased binding affinity toward their mRNA target relative to anequivalent natural polynucleic acid polymer. LNA modified polynucleicacid polymer can have increased binding affinity toward their mRNAtarget relative to an equivalent natural polynucleic acid polymer. ENAmodified polynucleic acid polymer can have increased binding affinitytoward their mRNA target relative to an equivalent natural polynucleicacid polymer. PNA modified polynucleic acid polymer can have increasedbinding affinity toward their mRNA target relative to an equivalentnatural polynucleic acid polymer. HNA modified polynucleic acid polymercan have increased binding affinity toward their mRNA target relative toan equivalent natural polynucleic acid polymer. Morpholino modifiedpolynucleic acid polymer can have increased binding affinity towardtheir mRNA target relative to an equivalent natural polynucleic acidpolymer. Methylphosphonate nucleotides modified polynucleic acid polymercan have increased binding affinity toward their mRNA target relative toan equivalent natural polynucleic acid polymer. Thiolphosphonatenucleotides modified polynucleic acid polymer can have increased bindingaffinity toward their mRNA target relative to an equivalent naturalpolynucleic acid polymer. Polynucleic acid polymer comprising 2′-fluoroN3-P5′-phosphoramidites can have increased binding affinity toward theirmRNA target relative to an equivalent natural polynucleic acid polymer.The increased affinity can be illustrated with a lower Kd, a higher melttemperature (Tm), or a combination thereof

In additional instances, a polynucleic acid polymer described herein maybe modified to increase its stability. In an embodiment where thepolynucleic acid polymer is RNA, the polynucleic acid polymer may bemodified to increase its stability. The polynucleic acid polymer may bemodified by one or more of the modifications described above to increaseits stability. The polynucleic acid polymer may be modified at the 2′hydroxyl position, such as by 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE),2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl(2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), or 2′-O-N-methylacetamido (2′-O-NMA) modification or by alocked or bridged ribose conformation (e.g., LNA or ENA). Thepolynucleic acid polymer may be modified by 2′-O-methyl and/or2′-O-methoxyethyl ribose. The polynucleic acid polymer may also includemorpholinos, PNAs, HNA, methylphosphonate nucleotides, thiolphosphonatenucleotides, or 2′-fluoro N3-P5′-phosphoramidites to increase itsstability. Suitable modifications to the RNA to increase stability fordelivery will be apparent to the skilled person.

A polynucleic acid polymer described herein can be constructed usingchemical synthesis and/or enzymatic ligation reactions using proceduresknown in the art. For example, a polynucleic acid polymer can bechemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the polynucleic acid polymer and target nucleicacids. Exemplary methods can include those described in: US5,142,047;US5,185,444; WO2009099942; or EP1579015. Additional exemplary methodscan include those described in: Griffey et al. , “2′-O-aminopropylribonucleotides: a zwitterionic modification that enhances theexonuclease resistance and biological activity of antisenseoligonucleotides,” J. Med. Chem. 39(26):5100-5109 (1997)); Obika, etal., “Synthesis of 2′-0,4′-C-methyleneuridine and -cytidine. Novelbicyclic nucleosides having a fixed C3, -endo sugar puckering”.Tetrahedron Letters 38 (50): 8735(1997); Koizumi, M. “ENAoligonucleotides as therapeutics”. Current opinion in moleculartherapeutics8 (2): 144-149 (2006); and Abramova et al. , “Noveloligonucleotide analogues based on morpholino nucleosidesubunits-antisense technologies: new chemical possibilities,” IndianJournal of Chemistry 48B:1721-1726 (2009). Alternatively, thepolynucleic acid polymer can be produced biologically using anexpression vector into which a polynucleic acid polymer has beensubcloned in an antisense orientation (i.e., RNA transcribed from theinserted polynucleic acid polymer will be of an antisense orientation toa target polynucleic acid polymer of interest).

A polynucleic acid polymer may be bound to any nucleic acid molecule,such as another antisense molecule, a peptide, or other chemicals tofacilitate delivery of the polynucleic acid polymer and/or target thenucleic acid to a specific tissue, cell type, or cell developmentalstage. The polynucleic acid polymer may be bound to a protein or RNA.The protein tethered to the polynucleic acid polymer may comprise asplicing factor to enhance, inhibit or modulate splicing and intronremoval. RNA tethered to the polynucleic acid polymer may comprise anaptamer or any structure that enhance, inhibit or modulate splicing andintron removal. The polynucleic acid polymer may be isolated nucleicacid.

A polynucleic acid polymer may be conjugated to, or bound by, a deliveryvehicle suitable for delivering the polynucleic acid polymer to cells.The cells may be a specific cell type, or specific developmental stage.The delivery vehicle may be capable of site specific, tissue specific,cell specific or developmental stage-specific delivery. For example, thedelivery vehicle may be a cell specific viral particle, or componentthereof, alternatively, the delivery vehicle may be a cell specificantibody particle, or component thereof. The polynucleic acid polymermay be targeted for delivery to beta cells in the pancreas. Thepolynucleic acid polymer may be targeted for delivery to thymic cells.The polynucleic acid polymer may be targeted for delivery to malignantcells. The polynucleic acid polymer may be targeted for delivery topre-malignant cells (that are known to develop into overt malignantphenotypes within a foreseeable future, such as pre-leukemias andmyelodysplastic syndromes or histopathologically defined precancerouslesions or conditions.

A polynucleic acid polymer may be conjugated to, or bound by, ananoparticle-based delivery vehicle. A nanoparticle may be a metalnanoparticle, e.g., a nanoparticle of scandium, titanium, vanadium,chromium, manganese, iron, cobalt, nickel, copper, zinc, yttrium,zirconium, niobium, molybdenum, ruthenium, rhodium, palladium, silver,cadmium, hafnium, tantalum, tungsten, rhenium, osmium, iridium,platinum, gold, gadolinium, aluminum, gallium, indium, tin, thallium,lead, bismuth, magnesium, calcium, strontium, barium, lithium, sodium,potassium, boron, silicon, phosphorus, germanium, arsenic, antimony, andcombinations, alloys or oxides thereof Sometimes a nanoparticle may beprepared from polymeric materials. Illustrative polymeric materialsinclude, but are not limited to, poly(ethylenimine) (PEI),poly(alkylcyanoacrylates), poly(amidoamine) dendrimers (PAMAM),poly(c-caprolactone) (PCL), poly(lactic-co-glycolic acid) (PLGA), orpolyesters (poly(lactic acid) (PLA). Sometimes a nanoparticle may befurther coated with molecules for attachment of functional elements. Insome cases, a coating comprises chondroitin sulfate, dextran sulfate,carboxymethyl dextran, alginic acid, pectin, carragheenan, fucoidan,agaropectin, porphyran, karaya gum, gellan gum, xanthan gum, hyaluronicacids, glucosamine, galactosamine, chitin (or chitosan), polyglutamicacid, polyaspartic acid, lysozyme, cytochrome C, ribonuclease,trypsinogen, chymotrypsinogen, a-chymotrypsin, polylysine, polyarginine,histone, protamine, graphene, ovalbumin or dextrin or cyclodextrin. Ananoparticle may include a core or a core and a shell, as in acore-shell nanoparticle. Sometimes, a nanoparticle may have at least onedimension of less than about 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm.

In some embodiments, a polynucleic acid polymer may be formulated with ananoparticle-based delivery vehicle for delivery to a site of interest(e.g., a malignant tissue site or a cell with deregulated proteinexpression). In some cases, a polynucleic acid polymer may be formulatedwith a nanoparticle-based delivery vehicle to facilitate and/or enabletransport across the blood-brain barrier (BBB).

Sometimes, a polynucleic acid polymer is coupled to a substance, knownin the art to promote penetration or transport across the blood-brainbarrier, e.g., an antibody to the transferrin receptor. In someembodiments, the polynucleic acid polymer is linked with a viral vector,e.g., to render the compound more effective or increase transport acrossthe blood-brain barrier. In some embodiments, osmotic blood brainbarrier disruption is assisted by infusion of sugars, e.g., mesoerythritol, xylitol, D(+) galactose, D(+) lactose, D(+) xylose,dulcitol, myo-inositol, L(−) fructose, D(−) mannitol, D(+) glucose, D(+)arabinose, D(−) arabinose, cellobiose, D(+) maltose, D(+) raffinose,L(+) rhamnose, D(+) melibiose, D(−) ribose, adonitol, D(+) arabitol,L(−) arabitol, D(+) fucose, L(−) fucose, D(−) lyxose, L(+) lyxose, andL(−) lyxose, or amino acids, e.g., glutamine, lysine, arginine,asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine,leucine, methionine, phenylalanine, proline, serine, threonine,tyrosine, valine, and taurine. Methods and materials for enhancing bloodbrain barrier penetration are described, e.g., in U.S. Pat. No.4,866,042, U.S. Pat. No. 6,294,520 and U.S. Pat. No. 6,936,589, eachincorporated herein by reference.

In one embodiment the polynucleic acid polymer may be bound to achemical molecule (e.g., non-peptide or nucleic acid based molecule),such as a drug. The drug may be a small molecule (e.g., having a MW ofless than 900 Da).

In one embodiment of the invention, the delivery vehicle may comprise acell penetrating peptide (CPP). For example, the polynucleic acidpolymer may be bound or complexed with a CPP. The skilled person willunderstand that any suitable CPP may be conjugated with the polynucleicacid polymer to aid delivery of the polynucleic acid polymer to and/orinto cells. Such CPPs may be any suitable CPP technology described byBoisguerin et al., Advanced Drug Delivery Reviews (2015), which isherein incorporated by reference. Suitable delivery vehicles forconjugation to the polynucleic acid polymer are also described inLochmann et al., ((European Journal of Pharmaceutics andBiopharmaceutics 58 (2004) 237-251), which is herein incorporated byreference).

The CPP may be an arginine and/or lysine rich peptide, for example,wherein the majority of residues in the peptide are either lysine orarginine. The CPP may comprise a poly-L-lysine (PLL). Alternatively, theCPP may comprise a poly-arginine. Suitable CPPs may be selected from thegroup comprising Penetratin; R6-Penetratin (“R6” disclosed as SEQ ID NO:120); Transportan; oligo-arginines; F-3; B-peptide; B-MSP; Pip peptides,such as Pip1, Pip2a, Pip2b, Pip5e, Pip5f, Pip5h, Pip5j; Pip5k, Pip51,Pip5m, Pip5n, Pip5o, Pip6a, Pip6b, Pip6c, Pip6d, Pip6e, Pip6f, Pip6g, orPip6h; peptide of sequence PKKKRKV (SEQ ID NO: 121); Penatratin; Lys4;SPACE; Tat; Tat-DRBD (dsRNA-binding domain); (RXR)4 (SEQ ID NO: 122);(RFF)3RXB (SEQ ID NO: 123); (KFF)3K (SEQ ID NO: 124); RgF2; T-cellderived CPP; Pep-3; PEGpep-3; MPG-8; MPG-8-Chol; PepFect6; P5RHH; R15(SEQ ID NO: 125); and Chol-R9 (“R9” disclosed as SEQ ID NO: 126); orfunctional variants thereof (e.g., see Boisguérin et al., Advanced DrugDelivery Reviews (2015)).

In one embodiment, the CPP comprises or consists of a Pip peptide. ThePip peptide may be selected from the group comprising Pip1, Pip2a,Pip2b, Pip5e, Pip5f, Pip5h, Pip5j; Pip5k, Pip51, Pip5m, Pip5n, Pip5o,Pip6a, Pip6b, Pip6c, Pip6d, Pip6e, Pip6f, Pip6g, and Pip6h.

In one embodiment of the invention, the delivery vehicle may comprise apeptide-based nanoparticle (PBN), wherein a plurality of CPPs (forexample one or more suitable CPPs discussed herein) form a complex withthe polynucleic acid polymer through charge interactions. Suchnanoparticles may be between about 50 nm and 250 nm in size. In oneembodiment the nanoparticles may be about 70-200 nm in size. In anotherembodiment the nanoparticles may be about 70-100 nm in size or 125-200nm in size.

In one embodiment, the polynucleic acid polymer may be complexed with adelivery vehicle, for example by ionic bonding. Alternatively, thepolynucleic acid polymer may be covalently bound to the deliveryvehicle. Conjugation/binding methods are described in Lochmann et al.,((European Journal of Pharmaceutics and Biopharmaceutics 58 (2004)237-251), which is herein incorporated by reference). For example, aconjugation method may comprise introducing a suitable tether containinga reactive group (e.g., —NH₂ or —SH₂) to the polynucleic acid polymerand to add the delivery vehicle, such as a peptide, post-syntheticallyas an active intermediate, followed by carrying out the couplingreaction in aqueous medium. An alternative method may comprise carryingout the conjugation in a linear mode on a single solid-phase support.

The delivery vehicle and polynucleic acid polymer may be thiol and/ormaleimide linked, such as thiol-maleimide linked. The conjugation of thepolynucleic acid polymer and the delivery vehicle may be byclick-chemistry, such as reaction of azido or 2′-O-propyargyl functionalgroups and alkyne groups on the respective molecules to be conjugated.In one embodiment, the delivery vehicle and polynucleic acid polymer maybe linked by a thioether bridge. In another embodiment, the deliveryvehicle and polynucleic acid polymer may be linked by a disulphidebridge. The skilled person will readily identify suitable linking groupsor reactions for conjugation of polynucleic acid polymer and thedelivery vehicle, such as a peptide.

In one embodiment the NSE repressor agent may comprise an SSO of thesequence cuucuaugcagccaaccuguagacu (SSO—NSE3) (SEQ ID NO: 53), or anucleic acid analogue thereof. In one embodiment the NSE repressor agentmay comprise an SSO of the sequence accuuuuucuucuaugcagccaac (SSO—NSE5)(SEQ ID NO: 54), or a nucleic acid analogue thereof. The skilled personwill note that NSE3 (cuucuaugcagccaaccuguagacu) (SEQ ID NO: 53) and NSE5(accuuuuucuucuaugcagccaac) (SEQ ID NO: 54) overlap in sequence. In oneembodiment, the NSE repressor agent may comprise an SSO having asequence of, or within, this overlapping sequence (i.e.accuuuuucuucuaugcagccaaccuguagacu) (SEQ ID NO: 55).

In one embodiment, the NSE repressor or activator agent comprises orconsists of any one SSO selected from the group comprising:

(SEQ ID NO: 18) aacuuaaagguuauaucuc (SSO A2); (SEQ ID NO: 19)uauaaauacgaauaaaucga (SSO A4); (SEQ ID NO: 21)caacacgacauaaccaaa (SSO A9); (SEQ ID NO: 23)aacauuucuauuuaguuaaaagc (SSO A11); (SEQ ID NO: 26)uuaguauuccuugacuuua (SSO A17); (SEQ ID NO: 32)gguaugagaacuauagga (SSO A23); (SEQ ID NO: 34)gguaauaagugucacaaa (SSO A25); (SEQ ID NO: 35)guaucauacauuagaagg (SSO A26); (SEQ ID NO: 37)gacugguaaauaauaaacauaauuc (SSO B2); (SEQ ID NO: 39)auauauuagagauacaucagcc (SSO B4); (SEQ ID NO: 45)uguggggugaccacagcuu (SSO B11); (SEQ ID NO: 51)uuagagaaucauuuuaaauaagac (SSO AN3);  and (SEQ ID NO: 56)cuguaaaagaaaauaga (PEkr),

or combinations thereof.

In another embodiment, the NSE activator agent comprises or consists ofany one SSO selected from the group comprising:

(SEQ ID NO: 18) aacuuaaagguuauaucuc (SSO A2); (SEQ ID NO: 19)uauaaauacgaauaaaucga (SSO A4); (SEQ ID NO: 21)caacacgacauaaccaaa (SSO A9); (SEQ ID NO: 32)gguaugagaacuauagga (SSO A23); (SEQ ID NO: 34)gguaauaagugucacaaa (SSO A25); (SEQ ID NO: 35)guaucauacauuagaagg (SSO A26); (SEQ ID NO: 45)uguggggugaccacagcuu (SSO B11);  and (SEQ ID NO: 56)cuguaaaagaaaauaga (PEkr),

or combinations thereof.

The NSE activator agent may comprise or consist of an SSO of thesequence aacuuaaagguuauaucuc (SSO A2) (SEQ ID NO: 18). The NSE activatoragent may comprise or consist of an SSO of the sequenceuauaaauacgaauaaaucga (SSO A4) (SEQ ID NO: 19). The NSE activator agentmay comprise or consist of an SSO of the sequence caacacgacauaaccaaa(SSO A9) (SEQ ID NO: 21). The NSE activator agent may comprise orconsist of an SSO of the sequence gguaugagaacuauagga (SSO A23) (SEQ IDNO: 32). The NSE activator agent may comprise or consist of an SSO ofthe sequence gguaauaagugucacaaa (SSO A25) (SEQ ID NO: 34). The NSEactivator agent may comprise or consist of an SSO of the sequenceguaucauacauuagaagg (SSO A26) (SEQ ID NO: 35). The NSE activator agentmay comprise or consist of an SSO of the sequence uguggggugaccacagcuu(SSO B11) (SEQ ID NO: 45).

In one embodiment the NSE-activator agent may comprise the SSO PEkrherein described. In one embodiment the NSE-activator agent may comprisean SSO of the sequence CUGUAAAAGAAAAUAGA (PEkr) (SEQ ID NO: 56). PEkrmay also be referred to as PEdel and it is understood that these termsare interchangeable.

In one embodiment, the NSE repressor agent comprises or consists of anyone SSO selected from the group comprising:

(SEQ ID NO: 53) cuucuaugcagccaaccuguagacu (SSO-NSE3); (SEQ ID NO: 54)accuuuuucuucuaugcagccaac (SSO-NSE5); (SEQ ID NO: 23)aacauuucuauuuaguuaaaagc (SSO A11); (SEQ ID NO: 26)uuaguauuccuugacuuua (SSO A17); (SEQ ID NO: 37)gacugguaaauaauaaacauaauuc (SSO B2); (SEQ ID NO: 39)auauauuagagauacaucagcc (SSO B4);  and (SEQ ID NO: 51)uuagagaaucauuuuaaauaagac (SSO AN3),or combinations thereof.

The NSE repressor agent may comprise or consist of an SSO of thesequence cuucuaugcagccaaccuguagacu (SSO—NSE3) (SEQ ID NO: 53). The NSErepressor agent may comprise or consist of an SSO of the sequenceaccuuuuucuucuaugcagccaac (SSO—NSE5) (SEQ ID NO: 54). The NSE repressoragent may comprise or consist of an SSO of the sequenceaacauuucuauuuaguuaaaagc (SSO All) (SEQ ID NO: 23). The NSE repressoragent may comprise or consist of an SSO of the sequenceuuaguauuccuugacuuua (SSO A17) (SEQ ID NO: 26). The NSE repressor agentmay comprise or consist of an SSO of the sequencegacugguaaauaauaaacauaauuc (SSO B2) (SEQ ID NO: 37). The NSE repressoragent may comprise or consist of an SSO of the sequenceauauauuagagauacaucagcc (SSO B4) (SEQ ID NO: 39). The NSE repressor agentmay comprise or consist of an SSO of the sequenceuuagagaaucauuuuaaauaagac (SSO AN3) (SEQ ID NO: 51).

In one embodiment the NSE repressor agent, such as an SSO, may bearranged to bind to guanine variant residue at rs4988000.

The skilled person will understand that combinations of two or more SSOsdescribed herein may be provided and/or used for treatment. For example,combinations of two, three, four, five or more NSE repressor agents maybe provided or combinations of two, three, four, five or more NSEactivating agents may be provided.

Where reference is made to reducing NSE inclusion in the mature RNA, thereduction may be complete, e.g., 100%, or may be partial. The reductionmay be clinically significant. The reduction/correction may be relativeto the level of NSE inclusion in the subject without treatment, orrelative to the amount of NSE inclusion in a population of similarsubjects. The reduction/correction may be at least 10% less NSEinclusion relative to the average subject, or the subject prior totreatment. The reduction may be at least 20% less NSE inclusion relativeto an average subject, or the subject prior to treatment. The reductionmay be at least 40% less NSE inclusion relative to an average subject,or the subject prior to treatment. The reduction may be at least 50%less NSE inclusion relative to an average subject, or the subject priorto treatment. The reduction may be at least 60% less NSE inclusionrelative to an average subject, or the subject prior to treatment. Thereduction may be at least 80% less NSE inclusion relative to an averagesubject, or the subject prior to treatment. The reduction may be atleast 90% less NSE inclusion relative to an average subject, or thesubject prior to treatment.

Where reference is made to increasing active-ATM protein levels, theincrease may be clinically significant. The increase may be relative tothe level of active-ATM protein in the subject without treatment, orrelative to the amount of active-ATM protein in a population of similarsubjects. The increase may be at least 10% more active-ATM proteinrelative to the average subject, or the subject prior to treatment. Theincrease may be at least 20% more active-ATM protein relative to theaverage subject, or the subject prior to treatment. The increase may beat least 40% more active-ATM protein relative to the average subject, orthe subject prior to treatment. The increase may be at least 50% moreactive-ATM protein relative to the average subject, or the subject priorto treatment. The increase may be at least 80% more active-ATM proteinrelative to the average subject, or the subject prior to treatment. Theincrease may be at least 100% more active-ATM protein relative to theaverage subject, or the subject prior to treatment. The increase may beat least 200% more active-ATM protein relative to the average subject,or the subject prior to treatment The increase may be at least 500% moreactive-ATM protein relative to the average subject, or the subject priorto treatment.

The terms active-ATM and functional-ATM may be used interchangeablyherein.

According to another aspect of the invention, there is provided use ofrs609261 genotyping to predict a subject response to therapy forconditions associated with ATM deregulation.

The conditions associated with ATM deregulation may comprise A-T orcancer.

In one embodiment, the presence of an rs609261 cytosine residue isassociated with a higher NSE activation, less efficient response of ATMto DNA double-strand break signaling, a higher cancer risk and lowersurvival relative to non-cytosine residue at the same position.

According to another aspect of the invention, there is provided acomposition comprising the NSE repressor agent of the invention herein.

According to another aspect of the invention, there is provided acomposition comprising the NSE activator agent of the invention herein.

In one embodiment, the composition is a pharmaceutically acceptableformulation.

The composition may comprise at least one other biologically activemolecule in addition to the polynucleic acid polymer. The biologicallyactive molecule may be drug or a pro-drug. The biologically activemolecule may comprise nucleic acid or amino acid. The biologicallyactive molecule may comprise a small molecule (e.g., a molecule of <900Daltons).

In some embodiments, pharmaceutical formulations described herein areadministered to a subject by an enteral administration route, by aparenteral administration route, or by a topical administration route.In some cases, pharmaceutical formulations described herein areadministered to a subject by an enteral administration route. In othercases, pharmaceutical formulations described herein are administered toa subject by a parenteral administration route. In additional cases,pharmaceutical formulations described herein are administered to asubject by a topical administration route.

Illustrative administration routes include, but are not limited to,parenteral (e.g., intravenous, subcutaneous, intramuscular,intra-arterial, intracranial, intracerebral, intracerebroventricular,intrathecal, or intravitreal), oral, intranasal, buccal, topical,rectal, transmucosal, or transdermal administration routes. In someinstances, the pharmaceutical composition describe herein is formulatedfor parenteral (e.g., intravenous, subcutaneous, intramuscular,intra-arterial, intracranial, intracerebral, intracerebroventricular,intrathecal, or intravitreal) administration. In other instances, thepharmaceutical composition describe herein is formulated for oraladministration. In still other instances, the pharmaceutical compositiondescribe herein is formulated for intranasal administration.

Pharmaceutical formulations described herein may include, but are notlimited to, aqueous liquid dispersions, self-emulsifying dispersions,solid solutions, liposomal dispersions, aerosols, solid dosage forms,powders, immediate release formulations, controlled releaseformulations, fast melt formulations, tablets, capsules, pills, delayedrelease formulations, extended release formulations, pulsatile releaseformulations, multiparticulate formulations, and mixed immediate andcontrolled release formulations.

Pharmaceutical formulations may include a carrier or carrier materialswhich may include any commonly used excipients in pharmaceutics andshould be selected on the basis of compatibility with the compositiondisclosed herein, and the release profile properties of the desireddosage form. Exemplary carrier materials include, e.g., binders,suspending agents, disintegration agents, filling agents, surfactants,solubilizers, stabilizers, lubricants, wetting agents, diluents, and thelike.

Pharmaceutically compatible carrier materials may include, but are notlimited to, acacia, gelatin, colloidal silicon dioxide, calciumglycerophosphate, calcium lactate, maltodextrin, glycerin, magnesiumsilicate, polyvinylpyrrollidone (PVP), cholesterol, cholesterol esters,sodium caseinate, soy lecithin, taurocholic acid, phosphotidylcholine,sodium chloride, tricalcium phosphate, dipotassium phosphate, celluloseand cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan,monoglyceride, diglyceride, pregelatinized starch, and the like.Liposomes can include sterically stabilized liposomes, e.g., liposomescomprising one or more specialized lipids. These specialized lipids canresult in liposomes with enhanced circulation lifetimes. Sometimes, asterically stabilized liposome can comprise one or more glycolipids oris derivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. See, e.g., Remington: The Science andPractice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack PublishingCompany, 1995); Hoover, John E., Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa. 1975; Liberman, H.A. and Lachman, L.,Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980;and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed.(Lippincott Williams & Wilkins 1999).

According to another aspect of the invention, there is provided a methodof treatment or prevention of functional-ATM protein deficiency in asubject, the method comprising identifying the presence of a non-thyminevariant residue rs609261 located at position -3 relative to the 3′splice site of NSE (cryptic exon in ATM intron 28) of the human genome,wherein the presence of a non-thymine variant residue rs609261 indicatesthat the subject has, or is susceptible to, functional-ATM proteindeficiency, and administration of an agent to the subject, which isarranged to replace the non-thymine variant residue rs609261 with athymine residue.

According to another aspect of the invention, there is provided a methodof treatment or prevention of functional-ATM protein deficiency in asubject, the method comprising replacing a non-thymine variant residuers609261 located at position −3 relative to the 3′ splice site of NSE(cryptic exon in ATM intron 28) of the human genome with a thymineresidue.

In one embodiment, replacing the non-thymine variant residue rs609261may comprise administration of an agent to the subject, which isarranged to replace the non-thymine variant residue rs609261 with athymine residue.

The agent for replacement of the non-thymine residue may be a genomicediting molecule, such as CRISPR-Cas9, or a functional equivalentthereof, together with an appropriate RNA molecule arranged to targetrs609261.

According to another aspect of the invention, there is provided a methodof treatment or prevention of functional-ATM protein deficiency in asubject, the method comprising identifying the presence of a guaninevariant residue at rs4988000 of the human genome, wherein the presenceof a guanine variant residue at rs4988000 indicates that the subjecthas, or is susceptible to, functional-ATM protein deficiency, andadministration of an agent to the subject, which is arranged to replacethe guanine variant residue at rs4988000 with adenine.

According to another aspect of the invention, there is provided a methodof treatment or prevention of functional-ATM protein deficiency in asubject, the method comprising replacing a guanine variant residue atrs4988000 of the human genome with an adenine residue.

In one embodiment, replacing the guanine variant residue at rs4988000may comprise administration of an agent to the subject, which isarranged to replace the guanine variant residue at rs4988000 with anadenine residue.

The agent for replacement of the guanine residue may be a genomicediting molecule, such as CRISPR-Cas9, or a functional equivalentthereof, together with an appropriate RNA molecule arranged to targetrs4988000.

According to a first aspect of the invention, there is provided a methodof screening a subject or a population of subjects for susceptibility tofunctional-ATM protein deficiency, wherein the screening comprisesdetermining the presence of a guanine variant residue at rs4988000 ofthe human genome, wherein the presence of a guanine variant residue atrs4988000 indicates that the subject (or group of subjects) has, or issusceptible to, functional-ATM protein deficiency.

According to another aspect of the invention, there is provided a methodof selecting a subject or a population of subjects for treatment orprophylaxis, wherein the subject is susceptible to functional-ATMprotein deficiency, the method comprising determining the presence of aguanine variant residue at rs4988000 of the human genome, wherein thepresence of a guanine variant residue at rs4988000 indicates that thesubject has, or is susceptible to, functional-ATM protein deficiency,and selecting such subject for treatment with an agent arranged toincrease functional-ATM levels in the subject.

According to another aspect of the invention, there is provided a methodof treatment or prevention of functional-ATM protein deficiency in asubject, the method comprising identifying the presence of a guaninevariant residue at rs4988000 of the human genome, wherein the presenceof a guanine variant residue at rs4988000 indicates that the subjecthas, or is susceptible to, functional-ATM protein deficiency, andadministration of an agent to the subject, which is arranged to increasefunctional-ATM levels.

The methods of the invention herein may comprise blocking a guaninevariant residue at rs4988000, for example using an SSO.

PE contains a natural DNA variant rs4988000 (G/A), which also influencesNSE recognition (FIG. 4H). Transfections of C and T minigenessystematically mutated at rs4988000 revealed that the rare A alleledecreased NSE inclusion on each pre-mRNA, both in U2AF35- andmock-depleted cells. Therefore, replacement of the guanine residue withadenine will decrease NSE inclusion, and increase the level offunctional ATM-protein.

The highest NSE inclusion is produced by the haplotype that is mostfrequent in Caucasians (CG), followed by haplotypes CA>TG>TA (referringtors609261 and rs4988000 respectively). Therefore, the methods andcompositions of the invention may be used in combination (concurrentlyor sequentially) to modify a CG haplotype to CA, TG, or TA. In oneembodiment, the methods and compositions of the invention may be used tomodify a CG haplotype to TA. In one embodiment, the methods andcompositions of the invention may be used to modify a CA haplotype to TGor TA. In one embodiment, the methods and compositions of the inventionmay be used to modify a CA haplotype to TA. In one embodiment, themethods and compositions of the invention may be used to modify a TGhaplotype to TA.

The methods and compositions of the invention may also be used incombination (concurrently or sequentially) to identify a CG haplotype ina subject, and optionally treat or select the patient according to theinvention. The methods and compositions of the invention may also beused in combination (concurrently or sequentially) to identify a CAhaplotype in a subject, and optionally treat or select the patientaccording to the invention. The methods and compositions of theinvention may also be used in combination (concurrently or sequentially)to identify a TG haplotype in a subject, and optionally treat or selectthe patient according to the invention.

According to another aspect of the invention, there is provided a methodof modifying regulation of NSE inclusion in a mature RNA transcript, themethod comprising the insertion or deletion of one or more splicingregulatory motifs upstream or downstream of the NSEs that compete withthe NSE for spliceosomal components, said regulatory motifs comprisingcryptic splice sites or pseudo-exons.

According to another aspect of the invention, there is provided a methodof modifying regulation of a functional protein expression, wherein thefunctional protein expression is regulated by NSE inclusion in a matureRNA transcript of the gene encoding protein, the method comprising theinsertion or deletion of one or more splicing regulatory motifs upstreamor downstream of the NSE that compete with the NSE for spliceosomalcomponents, said regulatory motifs comprising cryptic splice sites orpseudo-exons.

In one embodiment, the insertion or deletion of one or more splicingregulatory motifs is in genomic DNA of ATM intron 28.

The insertion of one or more splicing regulatory motifs may cause areduction in NSE inclusion in the mature RNA transcript The deletion ofone or more splicing regulatory motifs may cause an increase in NSEinclusion in the mature RNA transcript.

The insertion or deletion of one or more splicing regulatory motifs maycomprise the use of genome editing technology, such as CRISPR-Cas9.CRISPR-Cas9 may be provided with an appropriate targeting RNA molecule.

The subject or cells that are treated or screened according to theinvention may be mammalian. In one embodiment, the subject is a human.In one embodiment, the cells are human.

Kits and articles of manufacture are provided herein for use with one ormore methods described herein. The kits can contain one or more of thepolynucleic acid polymers described herein.

According to another aspect of the invention, there is provided a kitcomprising one or more oligonucleotide probes for identifying rs609261and/or rs4988000 variants.

The skilled person will be familiar with techniques for probing thepresence or absence of genetic sequence features. For example, theoligonucleotide probes may comprise primers for use in PCR amplifying aregion of a nucleic acid comprising rs609261 and/or rs4988000. Inanother embodiment the oligonucleotide probes may directly bind rs609261or rs4988000, wherein the binding may be detectable. The binding of theprobe may be detectable for example using SERS or SERRS technology.

The kits can also include a carrier, package, or container that iscompartmentalized to receive one or more containers such as vials,tubes, and the like, each of the container(s) comprising one of theseparate elements, such as the polynucleic acid polymers and reagents,to be used in a method described herein. Suitable containers include,for example, bottles, vials, syringes, and test tubes. The containerscan be formed from a variety of materials such as glass or plastic.

The articles of manufacture provided herein contain packaging materials.Examples of pharmaceutical packaging materials include, but are notlimited to, bottles, tubes, bags, containers, bottles, and any packagingmaterial suitable for a selected formulation and intended mode ofadministration and treatment.

A kit typically includes labels listing contents and/or instructions foruse, and package inserts with instructions for use. A set ofinstructions will also typically be included.

According to another aspect of the invention, there is provided a vectorcomprising the polynucleic acid polymer of the invention.

The vector may comprise a viral vector. The viral vector may compriseadeno-associated viral vector. The vector may comprise any virus thattargets the polynucleic acid polymer to malignant cells or specific celltype. Indications

In some instances, compositions and methods described herein are used totreat a genetic disorder or condition such as a hereditary disease.Compositions and methods described herein can be used to treat a geneticdisorder or condition such as a hereditary disease that is characterizedby an impaired production of a protein. Compositions and methodsdescribed herein can be used to treat a genetic disorder or conditionsuch as a hereditary disease that is characterized by a defectivesplicing.

Compositions and methods described herein can also be used to treat agenetic disorder or condition such as an autosomal dominant disorder, anautosomal recessive disorder, X-linked dominant disorder, X-linkedrecessive disorder, Y-linked disorder, mitochondrial disease, ormultifactorial or polygenic disorder. Compositions and methods describedherein can be used to treat an autosomal dominant disorder, an autosomalrecessive disorder, X-linked dominant disorder, X-linked recessivedisorder, Y-linked disorder, mitochondrial disease, or multifactorial orpolygenic disorder, in which the disorder or condition is characterizedby an impaired production of a protein. Compositions and methodsdescribed herein can also be used to treat an autosomal dominantdisorder, an autosomal recessive disorder, X-linked dominant disorder,X-linked recessive disorder, Y-linked disorder, mitochondrial disease,or multifactorial or polygenic disorder, in which the disorder orcondition is characterized by a defective splicing.

The condition associated with deregulated ATM expression may comprisecancer. Compositions and methods described herein can be used to treatcancer. In one embodiment the cancer comprises breast cancer. Cancer canbe a solid tumor or a hematologic malignancy. A solid tumor can be asarcoma or a carcinoma. Sarcoma can be a cancer of bone, cartilage, fatmuscle, vascular or hematopoietic tissues. Exemplary sarcoma can includealveolar rhabdomyosarcoma, alveolar soft part sarcoma, ameloblastoma,angiosarcoma, chondrosarcoma, chordoma, clear cell sarcoma of softtissue, dedifferentiated liposarcoma, desmoid, desmoplastic small roundcell tumor, embryonal rhabdomyosarcoma, epithelioid fibrosarcoma,epithelioid hemangioendothelioma, epithelioid sarcoma,esthesioneuroblastoma, Ewing sarcoma, extrarenal rhabdoid tumor,extraskeletal myxoid chondrosarcoma, extraskeletal osteosarcoma,fibrosarcoma, giant cell tumor, hemangiopericytoma, infantilefibrosarcoma, inflammatory myofibroblastic tumor, Kaposi sarcoma,leiomyosarcoma of bone, liposarcoma, liposarcoma of bone, malignantfibrous histiocytoma (MFH), malignant fibrous histiocytoma (MFH) ofbone, malignant mesenchymoma, malignant peripheral nerve sheath tumor,mesenchymal chondrosarcoma, myxofibrosarcoma, myxoid liposarcoma,myxoinflammatory fibroblastic sarcoma, neoplasms with perivascularepitheioid cell differentiation, osteosarcoma, parosteal osteosarcoma,neoplasm with perivascular epitheioid cell differentiation, periostealosteosarcoma, pleomorphic liposarcoma, pleomorphic rhabdomyosarcoma,PNET/extraskeletal Ewing tumor, rhabdomyosarcoma, round cellliposarcoma, small cell osteosarcoma, solitary fibrous tumor, synovialsarcoma, telangiectatic osteosarcoma.

Carcinoma can be a cancer developed from epithelial cells. Exemplarycarcinoma can include adenocarcinoma, squamous cell carcinoma,adenosquamous carcinoma, anaplastic carcinoma, large cell carcinoma,small cell carcinoma, anal cancer, appendix cancer, bile duct cancer(i.e., cholangiocarcinoma), bladder cancer, brain tumor, breast cancer,cervical cancer, colon cancer, cancer of Unknown Primary (CUP),esophageal cancer, eye cancer, fallopian tube cancer,gastroenterological cancer, kidney cancer, liver cancer, lung cancer,medulloblastoma, melanoma, oral cancer, ovarian cancer, pancreaticcancer, parathyroid disease, penile cancer, pituitary tumor, prostatecancer, rectal cancer, skin cancer, stomach cancer, testicular cancer,throat cancer, thyroid cancer, uterine cancer, vaginal cancer, or vulvarcancer. Hematologic malignancy is a malignancy of the blood system andcan include T-cell based and B-cell based malignancies. Exemplaryhematologic malignancy can include myeloid leukemia, myeloproliferativeneoplasias, peripheral T-cell lymphoma not otherwise specified(PTCL-NOS), anaplastic large cell lymphoma, angioimmunoblastic lymphoma,cutaneous T-cell lymphoma, adult T-cell leukemia/lymphoma (ATLL),blastic NK-cell lymphoma, enteropathy-type T-cell lymphoma,hematosplenic gamma-delta T-cell lymphoma, lymphoblastic lymphoma, nasalNK/T-cell lymphomas, treatment-related T-cell lymphomas, chroniclymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high riskCLL, non-CLL/SLL lymphoma, prolymphocytic leukemia (PLL), follicularlymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle celllymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma,extranodal marginal zone B cell lymphoma, nodal marginal zone B celllymphoma, Burkitt's lymphoma, non-Burkitt high grade B cell lymphoma,primary mediastinal B-cell lymphoma (PMBL), immunoblastic large celllymphoma, precursor B-lymphoblastic lymphoma, B cell prolymphocyticleukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma,plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B celllymphoma, intravascular large B cell lymphoma, primary effusionlymphoma, or lymphomatoid granulomatosis.

According to another aspect of the invention, there is provided a methodof a treatment or prevention of a disease pathology caused by an NSEinclusion in a pre-mRNA gene transcript comprising providing an agentarranged to bind to a cryptic splice site of a pseudoexon present on thepre-mRNA gene transcript, wherein the cryptic splice site is capable ofregulating inclusion of a nonsense-mediated RNA decay switch exon(NSE)into a mature RNA transcript of the gene.

Wherein the binding of the agent to the cryptic splice site of thepseudoexon present on the pre-mRNA gene transcript reduces the NSEinclusion.

The method may comprise a step of determining if a disease pathology iscaused by an NSE inclusion in a gene transcript prior to treatment.

The skilled person will understand that optional features of oneembodiment or aspect of the invention may be applicable, whereappropriate, to other embodiments or aspects of the invention.

EXAMPLES

Embodiments of the invention will now be described in more detail, byway of example only, with reference to the accompanying figures. Theseexamples are provided for illustrative purposes only and not to limitthe scope of the claims provided herein.

Abbreviations

NSE nonsense-mediated RNA decay switch exon in ATM intron 28

PE a 24-nt pseudoexon located 3′ of NSE in ATM intron 28

NMD nonsense-mediated RNA decay

A-T ataxia-telangiectasia

ATM gene deficient in ataxia-telangiectasia

SSO splice-switching oligonucleotide

DSB double-strand DNA break

DDR DNA damage response

MIR mammalian-wide interspersed repeat

BPS branch point sequence

PPT polypyrimidine tract

IR ionizing radiation

U2AF auxiliary factor of U2 small nuclear ribonucleoprotein

U2AF35 a 35-kD subunit of U2AF encoded by U2AF1

U2AF65 a 65-kD subunit of U2AF encoded by U2AF2

snRNA small nuclear RNAs

Example 1 SUMMARY

Phenotypic diversity and susceptibility to genetic disease is influencedby natural intronic variants, but their interactions with RNA-bindingproteins are largely unknown. Here a single-nucleotide polymorphism in adetained ATM intron was shown to gain functionality in cells lacking theauxiliary factor of U2 small nuclear ribonucloprotein (U2AF). Each U2AFsubunit was required for repression of a nonsense-mediated RNA decayswitch exon (NSE) in ATM intron 28. NSE was activated to a greaterdegree in the presence of cytosine than thymine at rs609261 located atposition −3 relative to the NSE 3′ splice site. The cytosine allele,which is predominant in Caucasians, resulted in a more efficientNSE-mediated inhibition of ATM expression than thymine, the principalallele in Asian populations. NSE activation was deregulated in leukemiccells and was influenced by the amino acid identity at U2AF35 residue34. Exploiting competition between NSE and a downstream pseudoexon,splice-switching oligonucleotides (SSOs) that repress or activate NSE tomodulate ATM expression were identified. Using RNA-Seq, U2AF-regulatedexon usage in the ATM signaling pathway was shown to be centered on theMRN/ATM-CHEK2-CDC25-cdc2/cyclin B axis and that U2AF preferentiallycontrols RNA processing of transcripts involved in cancer-associatedfusions and chromosomal translocations. These results reveal importantlinks between 3′ splice-site control and ATM-dependent response todouble strand DNA breaks, illustrate functional plasticity of intronicvariants in response to RNA-binding factors, demonstrate versatility ofSSOs to modify gene expression by targeting pseudo-splice sites inintrons and may explain ethnic differences in cancer risk and survival.

Introduction

Here, U2AF was shown to repress a nonsense-mediated decay (NMD) switchexon (NSE) in the ATM gene (ataxia-telangiectasia, A-T, mutated) andother proteins involved in 3′ss recognition that regulate NSE inclusionin mature transcripts were identified. The extent to which this eventlimits ATM expression depends on a common C/T variant rs609261 locatedin the NSE 3′ss consensus deep in intron 28. Also identified areintronic cis-elements that control NSE inclusion in mature transcriptsand splice-switching oligonucleotides (SSOs) that modulate NSEactivation by targeting a competing pseudoexon in the same intron. UsingRNA-Seq, it was next shown that the U2AF-mediatedregulation of DNAdamage response (DDR) pathway is centered on theATM-CHEK2-CDC25-cdc2/cyclin B axis, suggesting that it has coevolvedwith cellular responses to double-strand DNA breaks (DSBs). Finally, apreferential involvement of U2AF-regulated transcripts is demonstratedin cancer-associated gene fusions and chromosome translocations.

Results Identification of a U2AF-Repressed Cryptic Exon in ATM

It has been recently shown that depletion of each U2AF subunit resultedin down- and up-regulation of a large number of exons that werepredominantly alternatively spliced. When inspecting global RNAprocessing changes in cells depleted of U2AF35, an unexpectedly strongactivation of a cryptic, 29-nt ATM exon that was not annotated by RefSeq(termed NSE, FIG. 1A) was found. The NSE activation was observed also incells individually depleted of each U2AF35 isoform with isoform-specificsmall interfering RNAs (siRNAs) and with SSOs targeting 3′ss ofalternatively spliced U2AF1 exons Ab and 3, which encode isoform U2AF35band U2AF35a, respectively (FIG. 1A). Validation of RNA-Seq data usingRT-PCR showed that NSE was present in −10-20% of polyadenylatedtranscripts in untreated HEK293 cells, similar to levels observed inlymphoblastoid cell lines. The NSE inclusion levels increased to −75% incultures depleted of −90% U2AF35 and to −50% in cells depleted of −75%U2AF65 (FIG. 1B), were siRNA dose-dependent and inversely correlatedwith the amount of available U2AF heterodimers (FIG. 1C), consistentwith the requirement of each U2AF subunit for NSE repression. Inspectionof RNA-Seq data revealed retention of intronic sequences surrounding NSE(FIG. 1A), suggesting that intron 28 is ‘detained’ and could be splicedpost-transcriptionally. Retention levels of intron 28 were affectedneither by SSO- nor siRNA-mediated depletion of U2AF35 (FIG. 1A) and noother cryptic exon in this gene was activated to the same extent as NSE.Thus, NSE plays a key role in the exon-centric regulation of ATMexpression by U2AF.

NSE Activation and ATM Expression is Modified by rs609261

Examination of genomic sequences surrounding NSE revealed that position−3 relative to the NSE 3′ss is polymorphic (rs609261, FIG. 2A) in whichthymine (T) is predominant in African and Asian populations and cytosine(C) in Caucasians (FIG. 2A). The base identity at this position isimportant for universal exon recognition, with a CAG>TAG>AAG>GAGhierarchy of exon inclusion levels both at authentic andU2AF35-dependent 3′ss. To confirm that the NSE usage is allele-specific,splicing of two reporter constructs that contained C or T at thisposition was examined following transient transfections into humanembryonic kidney (HEK) 293 cells (FIG. 2B). The T construct yieldedlower NSE inclusion than the C reporter, both in untreated cells andcells individually depleted of each U2AF subunit (FIG. 2C).

To test whether the allele-specific NSE usage results in differentialprotein expression in cells lacking U2AF35, DNA was first sequenced fromavailable cell lines across rs609261 to obtain transfectable cellshomozygous for each allele. HEK293 cells were found to be homozygous forthe C allele and HeLa cells were homozygous for the T allele (FIG. 2D).Immunoblots from the U2AF35-depleted cells and untreated controlsconfirmed efficient depletion in each cell line and a greaterU2AF-mediated decline of ATM expression in the presence of the C allelethan the T allele (FIG. 2E,F). Depletion of UPF1, a key component of theNMD pathway, revealed a dose-dependent increase of NSE inclusion in ATMmature RNAs (FIG. 2G). No signal from a putative truncated ATM wasdetected on immunoblots from depleted cells.

Because U2AF-repressed and -activated exons show preferential responsesto U2AF-related proteins, HEK293 cells were depleted of PUF60 andCAPERa, and several heterogeneous nuclear RNPs, including hnRNP Al.PUF60 interacts with uridine-rich motifs at 3′ss and hnRNP Al forms aternary complex with the U2AF heterodimer on AG-containing U-rich RNAs.Depletion of either PUF60 or hnRNP Al increased NSE inclusion (FIG. 2H)while PUF60 overexpression led to NSE skipping (FIG. 2I). Thus, thers609261- and population-dependent NSE activation deep in ATM intron 28is regulated by U2AF, PUF60 and hnRNP Al, demonstrating howfunctionality of a common intronic polymorphism varies with cellularlevels of RNA-binding proteins that facilitate 3′ss recognition.

NSE Inhibition by SSOs Promotes ATM Expression

To test if NSE activation in cells lacking U2AF can be repressed torestore ATM expression, the C-allele reporter construct was individuallycotransfected with SSOs targeting each NSE splice site (FIG. 1A). SSOswere modified at each phosphorothioate linkage and 2′-O-methyl riboseand were designed to avoid the PPT of NSE, stable Mfold-predicted stemsand rs609261. Each SSO diminished NSE inclusion in a dose-dependentmanner both in exogenous (FIG. 3A) and endogenous (FIG. 3B) transcriptsand the SSO targeting the NSE 3′ss was more efficient than the SSObridging its 5′ss at the same concentrations.

Whether the NSE 3′ss SSO can increase ATM protein expression andactivation in cells exposed to ionizing radiation (IR) was nextexamined. The low ATM expression in cells lacking U2AF35 was partiallyrescued by this SSO, both in unexposed and IR-exposed cells (lanes 1 vs2 and 5 vs 6, FIG. 3C, lanes 5-8 vs 9-12, FIG. 8A) and the increase wasdose-dependent (FIG. 4D). Following IR, activated ATM autophosphorylatedat S1981 showed reduced signal in depleted cells as compared tountreated cells (lane 6 vs 8, FIG. 3C, and lanes 1-4 vs 5-8, FIG. 8A).Exposure to the NSE 3′ss SSO slightly increased also activated ATM(lanes 5-8 vs 9-12, FIG. 8A, lane 5 vs 6, FIG. 3C). To begin to exploreputative effects of SSO-mediated NSE repression on ATM signaling, wildtype CHEK2 was also overexpressed in (mock)irradiated cells(mock)depleted of U2AF (FIG. 8A). CHEK2 is a serine/threonine kinasephosphorylated by ATM at T68 in response to DNA double-strand breaks(DSBs). However, cells lacking U2AF had markedly lower levels ofendogenous CHEK2 compared to controls, which did not appear to bealtered by the NSE 3′ss SSO (lanes 1-4 vs 5-8 vs 9-12) whereas exogenousCHEK2 was increased in depleted cells both in IR-exposed and—unexposedcells (lanes 1-4 vs 5-8, see also FIG. 5 and FIG. 8B,C further below).

Taken together, NSE activation was efficiently inhibited by SSOs thatblock access to NSE splice sites and do not support RNase H cleavage.The more efficient SSO partially rescued the NSE-mediated inhibition ofATM.

Activation of a NMD Switch Exon is Influenced by a Downstream Pseudoexon

To identify intronic regulatory cis-elements that control NSE inclusionin mature transcripts, a previously reported A-T mutation IVS28-159A>Gwas utilized. This mutation was observed to activate the NSE 3′ss whilerepressing its 5′ss and promoting a downstream 5′ss instead, introducinga 112-nt cryptic exon in the mRNA. There is a strong 3′ss consensuspreceded by optimal BPS/PPT motifs observed within this exon, which maybind U2AF and activate a smaller, 24-nt pseudoexon (termed PE; FIG. 4A).Examination of published RNA crosslinking/immunoprecipitation data inATM showed U2AF65 binding upstream and downstream of NSE and upstream ofPE, suggesting that NSE activation may be controlled by competitionbetween partially productive spliceosomes assembled at the PE 3′ss andthe NSE 3′ss. The two 3′ss are conserved in mammals but are separated bya distance smaller than the minimal size of human introns, stericallypreventing simultaneous recognition of NSE and PE (FIG. 4A). Inagreement with this hypothesis, deletion of the PE PPT/3′ss introducedin the C minigene, which should alleviate NSE repression throughdiminished U2AF binding to PE, increased NSE inclusion (FIG. 4B). Thisdeletion also brought about retention of the intron that separates NSEand PE, mimicking the splicing pattern of the A-T mutation IVS28-159A>G.Increasing the intron length from 59 to 79 nt, thereby overcoming asteric hindrance imposed by the insufficient distance between the twopseudo-3′ss, also improved NSE inclusion and diminished the intronretention (FIG. 4B).

To test if NSE inactivation can influence PE inclusion in mRNA, the NSE3′ss was first eliminated. This mutation activated a cryptic 3′ss 7-ntdownstream of the authentic NSE 3′ss (lanes 1, 2 and 6, 7, FIG. 4C, FIG.21). This cryptic 3′ss showed a diminished requirement for U2AF. Becauseextending the intron length between NSE and PE on this background failedto activate PE (FIG. 4C, lanes 3 and 8) and PE lacks exonic splicingenhancers and has a suboptimal BPS (FIG. 22), a 24-nt stem loop derivedfrom a mammalian-wide interspersed repeat (MIR) was inserted in themiddle of PE. This MIR hairpin acts as a nearly universal exondefinition module through an exposed splicing enhancer in a terminal RNAtriloop. The enlarged PE was strongly activated in mock-depleted cells,but was outcompeted by NSE in cells lacking U2AF35 (lanes 4 and 9),indicating that NSE inclusion is more dependent on U2AF35 than PE. Theconstruct containing both the MIR insertion in PE and the extendedintron finally generated mRNAs containing both NSE and PE (lanes 5 and10).

Intronic SSOs Targeting Competing Pseudoexons to Modulate GeneExpression

Next, the MIR reporter was employed to test the impact of NSE and PESSOs on exon usage and ATM expression. FIG. 4D shows that the NSE 3′ssSSO repressed transcripts containing NSE and upregulated those with PEwhereas the opposite effect was found for SSOs targeting the MIRenhancer loop in PE. The same pattern was observed for the reporter inwhich NSE and PE were separated by a distance insufficient for theirsimultaneous inclusion in mRNA (FIG. 4E). These results suggested thatSSOs targeting PE and/or U2AF65 binding sites upstream of PE maypotentially promote NSE inclusion and reduce ATM expression while theNSE SSOs should have the opposite effect. This approach would provide abroad strategy to modulate gene expression in either direction byantisense-based targeting of competing pseudoexons, one of which iscritical for gene regulation. To test this concept, SSOs targeting PE3′ss and 5′ss were examined. Although each PE SSO induced NSE skipping,both on exogenous and endogenous transcripts (FIG. 4F), SSOs targetingU2AF65 binding sites just upstream of PE (FIG. 4A), i.e. theNSE-repressing sequence (construct de1PPT/AG, FIG. 4B), reduced PEinclusion and slightly increased NSE in the MIR reporter (FIG. 4G). Incontrast, a longer oligo extended in the 5′ direction (SSO-PEBP, FIG.20) did not show any effect.

PE contains a natural DNA variant rs4988000 (G/A), which may alsoinfluence NSE recognition (FIG. 4H). Transfections of C and T minigenessystematically mutated at rs4988000 revealed that the rare A alleledecreased NSE inclusion on each pre-mRNA, both in U2AF35- andmock-depleted cells. Thus, the highest NSE inclusion was produced by thehaplotype that is most frequent in Caucasians (CG), followed byhaplotypes CA>TG>TA.

Taken together, the haplotype-dependent activation of the U2AF-repressedNSE can be modified by SSOs that target U2AF65 intronic binding sitesupstream of competing pseudo-3′ss, potentially providing a generalmethod to manipulate exon-centric gene expression by antisense-basedtargeting of NMD switch exons and their regulatory motifs in introns.

Regulation of ATM Signaling by U2AF: DSBs at the Focal Point

Because ATM is a key apical kinase in the DDR pathway and NMD switchexons often regulate genes encoding protein interaction partners,U2AF35-induced RNA processing changes of currently known ATM substratesand other constituents of the ATM signaling network were systematicallycharacterized. Interestingly, although genes involved in the DDR andcell cycle control that contained U2AF35-dependent exons were onlymarginally enriched (FDR=0.08), each component in theATM-CHEK2-CDC25-CDC2/cyclin B axis showed RNA processing alterations(FIG. 5A, FIG. 9). This pathway is critical for ATM signaling of DSBs.

First, reduced ATM expression in cells lacking U2AF (FIG. 8) wasassociated with decreased CHEK2 mRNA, increased retention of CHEK2intron 10, and skipping of exons 9 and 11 (FIG. 5A). RNA processingalterations of known CHEK2 substrates were limited to genes regulatingthe cell cycle (CDC25A, CDC25B, CDC25C and TTK; FIG. 5A, S3A-B, 11A) andwere not apparent in genes involved in DNA repair (BRCA112, XRCC1,FOXM1, TRIM28) or p53 signaling (TP53, MDM4, CABIN1 , STRAP, AATF).CHEK2 exon 9 skipping, which would be predicted to activate NMD, wasonly marginally increased 24 hrs after IR and did not contribute to thedecline of total CHEK2 observed as early as 30 min after IR (FIGS. 5Band 5C). As CHEK2 exon 9 inclusion was increased only for the highestconcentration of UPF1 siRNAs (FIG. 5D), HEK293 cells were transcfectedwith an SSO targeting its 3′ss. This treatment induced exon 9 skippingand reduced expression of the CHEK2 protein, however, it also increasedNSE activation (FIG. 5E). In contrast, SSOs targeting NSE or PE did nothave any effect on CHEK2 exon 9 inclusion (FIG. 5F). Exon 9 skipping,but not NSE, was also dramatically increased in cells lacking SF3B1(FIG. 5G). To address why exogenous expression of CHEK2 was increased incells lacking U2AF35 as compared to controls (FIG. 8A), HEK293 cellswere cotransfected with the CHEK2-repressing SSO and a CHEK2-expressingplasmid (FIGS. 8B, and 8C). Reduced endogenous CHEK2 was associated witha significant increase of exogenous CHEK2 also in U2AF-proficient cells,pointing to a tight homeostatic regulation of the total CHEK2 protein inthe cell.

Second, U2AF was required for full activation of CDC25A exon 6 (FIG.5A), which encodes a residue (S178) that is phosphorylated by CHEK2 andCHEK1, facilitating binding of 14-3-3. U2AF35 was also required forinclusion of exon 3 of CDC25B and CDC25C (FIGS. 10A and 10B), confirmingprevious microarray data. CDC25B exon 3 encodes multiple phosphorylatedresidues, including a B-domain residue S169, phosphorylated by MAPKAPkinase 2 and pEg3. This isoform localizes to the centrosomes andaccumulates during mitosis. CDC25C exon 3 encodes T67 phosphorylated bycdc2/cyclin B as a part of the auto amplification loop. PhosphorylatedT67 inCDC25C creates a binding site recognized by the WW domain of PIN1,which sustained activation of a U2AF-repressed NMD switch exon (FIG.11B), possibly modifying catalytic activity of this abundantpeptidyl-prolyl isomerase. Finally, cyclin B1 and B2 mRNAs wereupregulated in cells lacking U2AF35 as well as cyclin B1-interactingprotein (CCNB11P1, also known as HEI10), although their RNA processingpattern did not appear to be altered (FIG. 5A). Cyclin B upregulationwas associated with a detained CDK1 intron (FIG. 11C), which may bespliced post-transcriptionally.

ATM recruitment to DSB is facilitated by the MRN complex, consisting ofMRE11, RAD50 and NBN. NBN showed no obvious RNA processing changes incells lacking U2AF35, but RAD50 mRNA was downregulated, possibly throughactivation of a NMD switch exon and/or additional splicing alterations(FIG. 12A-C and FIG. 9). The last MRE11A exon was upregulated as aresult of a promotion of distal alternative polyadenylation site indepleted cells, which is present in most cell types, but not in B cells.DEXSeq analysis did not detect significant RNA processing changes intranscripts encoding other members of the phosphatidylinositol 3kinase-like family of serine/threonine protein kinases (ATR and PRKDC),nor in BRCA1/2, RNF 168 and the ATM interactor ATMIN. Additional ATMinteracting partners with altered exon or gene expression included RPS6,SRSF 1 and other SR proteins, EP300, RPA2, BLM, FANCD2 and FANCI,PPP2R5C and PPP2R5D, and SMC3, a central component of the cohesincomplex (FIG. 9).

Depletion of U2AF35 was associated with preferential alterations ofgenes/exons involved in chromatin modification, which have numerousfunctional links to ATM signaling (FIG. 9). For example, the INO80chromatin remodeling complex is phosphorylated by ATM and isfunctionally linked to checkpoint regulators, including CHEK2. U2AFinhibited INO80C isoforms containing 54-nt exons that encode peptidesthat are absent in the yeast Ies6 homolog, which is critical to INO80function in vivo and is likely to alter heterodimer formation with ACTR5and nucleosome binding. Expression of multiple components of the INO80complex was altered in depleted cells, including ACTR5, ACTL6A andRUVL2B. Many INO80 subunits localize preferentially in telomeres andtheir mutations result in telomere elongation. U2AF is required for fullinclusion of TERF 1 exon 7 in mRNA (FIG. 13A), regulating the abundanceof TRF 1 (exon 7+)/PIN2 (exon 7-) isoforms, important components of theshelterin complex. Exon 7 encodes multiple phosphorylated serineresidues and both isoforms can heterodimerize through the dimerizationdomain. TRF1 binding to telomeres is promoted by ATM inhibition whereasATM-mediated phosphorylation impairs TRF 1 interaction with telomericDNA. TRF1 association with telomeres is also negatively regulated byRAD50. TRF1-interacting TIF2 is another shelterin protein localized innuclear matrix and encoded by TINF2. TIF2 exists in at least twoisoforms produced by alternative splicing, termed TIN2S and TIN2L. TIN2Lcontains an extra NM binding domain and associates more strongly withthe nuclear matrix than TIF2S,which is encoded by a transcript withretained 3′ introns that form a long 3′ untranslated region. This mRNAisoform was repressed by U2AF (FIG. 13B).

Collectively, these results show that theMRN/ATM-CHEK2-CDC25-cdc2/cyclin B axis is at the center of theU2AF35-mediated control of DDR, although the U2AF regulation extendsinto additional ATM substrates involved in chromatin modification andtelomere length control.

U2AF Preferentially Controls RNA Processing of Transcripts Involved inLeukemia-Associated Fusions

CHEK2 phosphorylates PML (Promyelocytic Leukemia) and appears to requirePML for subsequent autophosphorylation. Depletion of U2AF35 promoted theuse of proximal alternative polyadenylation site of PML, leading to theupregulation of the shortest PML isoform, which lacks the last exoncoding for the nuclear export signal (FIG. 14A). The long and short PMLisoforms have distinct functions; for example, nuclear PML isoforms, butnot the cytoplasmic isoform, are positive regulators of IFNγ signaling.The C-terminus of the longest PML isoform specifically interacts withAML1 to enhance AML1-mediated transcription, suggesting that U2AFdeficiency could impair PML-AML1 interactions. PML also binds PIN1 andthis interaction promotes PML degradation in a phosphorylation-dependentmanner. U2AF depletion increased a PINI NMD exon (FIG. 11B), potentiallylimiting expression of this highly abundant peptidyl-prolyl isomerase,which interacts with many phosphoproteins to regulate mitosis, includingphosphorylated CDC25.

Apart from PML, U2AF35 depletion upregulated other RARA partners,including NPM1 (FIG. 14B). This event was associated with promotion of aproximal polyadenylation site, thus increasing the abundance of shorter,presumably more stable transcripts. An alternatively spliced exon ofBCOR, a BCL6 corepressor that forms BCOR-RARA fusions and interacts withseveral histone deacetylases to increase BCL6 transcriptionalrepression, was also downregulated (FIG. 14C).

Interestingly, the overlap between U2AF35-sensitive genes/exons and1,187 genes involved in cancer-associated gene fusions and 300 genesinvolved in recurrent chromosome translocations was greater thanexpected, with more significant P values observed for genes withdifferentially used exons than those implicated by Cufflinks at thetranscript level (Table 1). Similarly, sharing of genes frequentlymutated in the myelodysplastic syndrome and genes differentiallyexpressed upon U2AF35 depletion was significantly higher than expected(P<0.01, hypergeometric test). Thus, RNA processing of transcriptsinvolved in cancer-associated gene fusions and chromosome translocationsis preferentially regulated by U2AF.

To test the function of cancer-associated U2AF1 mutations in NSEsplicing, reconstitution experiments were performed with wild-type andmutated U2AF35 constructs that were cotransfected with the C minigeneinto cells (mock)-depleted of U2AF35 (FIG. 6). NSE activation wasrepressed by either U2AF35 isoform to a similar extent, as well asU2AF35a containing substitutions of S34 in the zinc finger 1 domain, themost frequently mutated U2AF35 residue in cancer. In contrast, only apartial rescue was achieved by substitutions of Q157 in the second zincfinger domain where these mutations are less frequent. Other S34mutations failed to fully reconstitute the defect, including S34T andsubstitutions with small amino acids, although a large residue at thisposition (S34R) was efficient. Thus, the identity of the residue atposition 34 of U2AF35 is important for NSE recognition.

Finally, a low degree of NSE activation was detected in diverse humantissues, both in hexamer-primed samples and polyadenylated transcripts(FIG. 15A). The proportion of NSE-containing RNAs was on average higherin leukemic cells than in normal cells, with some samples exhibitingvery high levels not observed in normal tissues (FIGS. 15B and 15C),potentially contributing to reduced ATM expression previously observedboth in leukemias and solid tumors. NSE inclusion was also examined inpolyadenylated RNAs extracted from a panel of lymphoblastoid cell linesexposed to cold and heat shock at the indicated temperatures prior tolysis (FIGS. 15D and 15E). Interestingly, NSE appeared to be activatedto a minor extent by exposing cells to 42° C. but not atsubphysiological temperatures (FIG. 15D), suggesting that markedlyhigher NSE inclusion levels in malignant cells are unlikely to beexplained by a cold shock encountered during storage of patients'samples. Since proteomic profiling of Jurkat cells exposed to a heatstress at 43° C. revealed diminished expression of several proteinsincluding U2AF35a, these results further support U2AF35 as a specificNSE repressor.

Discussion

The work described herein significantly expands currently known linksbetween RNA processing and DDR pathways (FIGS. 5 and 9). An alternativesplicing-coupled NMD switch exon critical for ATM expression wasidentified (FIGS. 1 and 3) and its importance in cancer risk wasexamined (FIG. 2, FIGS. 6 and 15). How intronic haplotypes influenceinclusion of this exon in mature transcripts and their functionaldependence on cellular levels of RNA-binding proteins involved in 3′ssselection was also shown (FIGS. 2 and 4H). Finally, SSOs were identifiedthat modulate activation of this exon by targeting its regulatorysequences and propose a novel antisense strategy to modify geneexpression.

U2AF is an important 3′ss recognition complex and a critical regulatorof alternative splicing. In addition to expanding protein-proteininteractions, alternative splicing has evolved to fine-tune quantitativegene expression through NMD, in agreement with alterations of many NMDexons in cells lacking this factor (FIGS. 1, 5 and 13). Peptides encodedby alternatively spliced exons are enriched in disordered regions andpost-translation modification (PTM) sites, which are required fordynamic and reversible switching between two or more isoforms.Conversely, PTMs regulate numerous splicing factors, including proteinsinvolved in NSE regulation. This complexity represents a clear challengeahead and can be exemplified by the observed NSE activation upontargeting of CHEK2 exon 9 (FIG. 5E). Reduced CHEK2 expression may alterinteractions with other kinases such as CDK11, which is constitutivelyphosphorylated at S737 in a CHEK2-dependent manner and interacts withU2AF65 and PUF60 , creating a regulatory loop that controls NSE levels(FIG. 2H,I).

These results suggest that U2AF is an integral part of the DDR control,contributing to fine-tuning of its PTM network and subject to PTMsitself. U2AF35 was found among proteins that showed increasedphosphorylation at S59 upon DNA damage. This serine residue is presentonly in U2AF35a and is replaced by alanine in U2AF35b. Exogenousexpression of U2AF35b was higher than U2AF35a and the relative abundanceof U2AF35b increased upon depletion of U2AF65, suggesting that the twoU2AF35 isoforms may differentially interact with U2AF65 and may not haveequivalent roles in DDR. However, U2AF35- and U2AF65-regulated exonsvastly overlap and most, but not all, RNA processing changes found inU2AF35 depleted cells are attributable to the lack of the U2AFheterodimer, including the NSE activation (FIG. 1C).

U2AF-repressed exons have a distinct 3′ss organization and response toU2AF-related proteins as compared to U2AF -activated exons, suggestingthat the exon repression involves direct RNA binding. This is supportedby the observed NSE activation on exogenous transcripts that do notundergo NMD and by the SSO-induced NSE blockage (FIGS. 2 and 4). NSElacks AG dinucleotides between the predicted BPS and 3′ss, its AGexclusion zone is longer than the average and has an unusual stretch of5 conserved guanines upstream of the BPS, which may contribute to stablesecondary structures across 3′ss that might be required for therepression. The adenine-rich 3′ portions of both NSE and PE are moreconserved in evolution than their 5′ parts (FIG. 4A), potentiallyproviding important ligand interactions, given the propensity of adenineto occupy unpaired positions in structured RNAs. Interestingly, primateNSEs have uridine at position −3 and longer PPT than lower mammals,which have cytosine at this position. Although direct RNA bindingappears to be the simplest explanation for exon repression by U2AF,U2AF35 depletion led to downregulation of several proteins involved inNMD (Table S4), which may contribute to NSE activation on endogenoustranscripts. In addition, physical interactions between U2AF65 and theC-terminus of TRF1 or other components of the ATM signaling network mayalso participate in NSE regulation.

Apart from U2AF1/U2AF2, additional genes involved in 3′ss selection havebeen found mutated in cancer. Interestingly, chronic lymphocyticleukemias with SF3B1 mutation were associated with a cryptic 3′ssactivation of ATM exon 46, leading to ATM truncation. Recently, splicingof an EZH2 exon as a result of cancer-associated SRSF2 mutation wasimplicated in impaired hematopoietic differentiation and the same NMDexon was upregulated also upon U2AF35 depletion (FIG. 12D). Whetherthese exons are targets of a common 3′ss recognition pathway underlyingleukemogenesis remains to be established. In contrast, NSE inclusion didnot appear altered in cells depleted of SF3B1, which produced almostcomplete skipping of CHEK2 exon 9 (FIG. 5G).

Because NSE activation may restrict ATM expression both in normal andcancer cells (FIGS. 1, 2, and 15) and ATM is a limiting factor in theDDR pathway, cytosine at rs609261 may confer a relative ATM deficiencynot only in (pre-)malignant cells but also in the germline. ATM kinaseactivity appears to be a good predictor of A-T severity, however, thediversity of A-T alleles does not fully account for the spectrum ofclinical symptoms. Genes involved in NSE activation (FIG. 1, 2) mightcontribute to clinical heterogeneity of A-T patients, particularly thosewith ‘leaky’ mutations. Natural variants modifying NSE inclusion (FIGS.2C-F and 4H) may also contribute to the phenotypic complexity of A-T oreven A-T heterozygotes that lack overt clinical features but may displayincreased radiosensitivity and cancer risk, consistent with the centralfocus of U2AF-regulated exon usage within the ATM signaling network(FIG. 9).

These results predict that NSE activation is on average more efficientin Caucasians than in Asian populations as a result of a higherfrequency of the C allele at rs609261 in the former (FIG. 2A). AsianAmericans have lower mortality rates for common malignancies thanCaucasians that persist over a long-period of time. The risk ofhematopoietic malignancies also varies greatly by ethnic group and theirincidence is the highest in white populations, including non-Hodgkin andHodgkin lymphomas, which are associated with A-T. This trend alsopersists in migrants and continues in subsequent generations. Althoughlymphoblastoid leukemias, lymphomas and other cancer types show distinctincidence rates across Asian and Caucasian populations, no significantethnic differences in the age-standardized incidence rates were foundfor myeloid leukemias, which does not appear to be more prevalent inA-T, unlike lymphoid malignancies or other cancers. Asian cancerpatients respond more favorably than Caucasian patients to cytotoxictherapy and have on average a longer median survival. Asian cancerpatients were also reported to have a lower prevalence of some genefusions than Caucasians, potentially reflecting their capacity torespond to DSBs. rs609261 showed the lowest p-value of ATM variants inCochrane-Armitage tests of association with glioma. rs2235006 (ATMallele F582L), which is located only ˜35 kb upstream of rs609261 in aregion of minimal recombination, was associated with a high risk (OR11.2) of chronic lymphocytic leukemia. This study genotyped 1467 codingnonsynonymous SNPs in 865 candidate genes and implicated variants ingenes encoding the ATM-BRCA2-CHEK2 DDR axis as the most significant riskpathway. Allelic association studies of nonagenarians/centenarians andyounger controls also suggested association between ATM and longevity.Finally, ethnic differences were noted also for mutation rates in genesfrequently altered in hematological malignancies; for example, SF3B1mutations in chronic lymphocytic leukemias were less frequent in Chinesethan in European populations.

Although these considerations collectively support the importance ofrs609261-dependent NSE activation in cancer risk and survival, the U2AF-and hnRNP A1-dependency of NSE inclusion (FIG. 2H, S8B) demonstratesthat it is by no means fixed. Variable expression patterns of theseproteins from one malignancy to another would imply a ‘capriciousfunctionality’ of this variant. Many more polymorphic sites with thisattribute are likely to be established in future, contributing not onlyto the inter-individual variability of gene expression throughrestrictive capacity of ‘poison’ cryptic exons, but potentially also tothe ‘missing heritability’ of complex traits and failures of genome-wideassociation studies, particularly in cancer.

Although RNA-Seq is a powerful tool to examine global transcriptome inresponse to DNA damage, rigorous standards that correctly estimatebiological and statistical significance of the observed alterations inRNA processing are yet to be implemented. Given a high stringency of theDEXSeq algorithm, the existence of additional biologically important RNAprocessing events responsive to U2AF cannot be excluded. For example,upregulation of a proximal polyadenylation site in CHEK1, which wascoupled with upregulation of 24-nt and 27-nt exons in CLASP1, wouldimplicate the ATM apoptotic pathway. These events were not detected byDEXSeq but were see genomic browsers and require confirmation. Theapoptotic pathways are of particular interest in the myelodysplasticsyndrome which shows susceptibility of myeloid progenitors to theprogrammed cell death and where deregulation of genes involved in ATMsignaling was found in more advanced but not initial clinical stages.Interestingly, U2AF1 mutations were also found to be more frequent inadvanced stages and were associated with shorter survival. This studyalso highlights current limitations of incomplete transcript annotationand the importance of examining cryptic exons in RNA-Seq data. FutureRNA-Seq studies should therefore attempt at global detection of NMDevents associated with alternative splicing, which has been hindered bythe instability of stop codon-containing transcripts.

Finally, this study demonstrates efficient repression of a key NMDswitch exon in ATM by SSOs that also increased ATM protein levels (FIG.3A-D, FIG. 8). It also reveals competing regulatory motifs of NSE in thesame intron (FIG. 4A-C, H) that could be exploited as a target forSSO-mediated modulation of gene expression (FIG. 4D-G). This approachcan be combined with genome-editing such as CRISPR-Cas9 to delete orintroduce splicing regulatory motifs or protein binding sites implicatedby minigene studies (FIG. 4C) and should also help us to find efficientintronic SSOs with desired outcomes for RNA processing. The search forsuch SSOs is more challenging than for those targeting human exons. Forexample, most SSOs systematically covering SMN2 exon 7 stimulated exonskipping, an event exploited for treatment of spinal muscular atrophy,however, −20% induced exon inclusion. By analogy, the desiredstimulation of intron splicing was found only for 10% of SSOs targetingINS intron 1 while the majority failed to show this effect. The proposedstrategy takes advantage of a much higher information content of humanauxiliary splicing sequences as compared to lower organisms and shouldbe greatly facilitated by future global pre-mRNA folding studies. Forexample, unlike the SSO that efficiently blocked the NSE 3′ss (SSO-NSE3,FIG. 3A,B), a partially overlapping morpholino extending only 7-nt intoNSE failed to repress the same 3′ss to rescue splicing of mutationIVS28-159A>G, despite targeting U2AF binding sites (FIG. 4A). Thissuggests that the morpholino oligo may have blocked access to structuresor motifs that are not responsible for exon activation, but exonrepression, in agreement with these finding (FIG. 1A-C). Administrationof antisense-based RNA processing activators or inhibitors that targetor avoid binding sites of splicing factors in introns could be exploitedtherapeutically to shape beneficial or detrimental consequences of NMDin cancer cells. This approach is supported by a broad recognition thatNMD serves primarily a regulatory function across a wide range oftranscripts and may also promote translation of NMD substrates thatproduce truncated polypeptides, which may stimulate anti-tumor immunity.

Materials and Methods Plasmid Constructs

ATM minigenes were prepared by cloning −0.9-kb amplicons into XhoI/XbaIsites of the U2AF1 construct. Cloning primers are shown in Table 51.Full inserts were sequenced to confirm the identity of intended changesand exclude undesired mutations. PUF60 expression vectors were alsoused. The hnRNP A1 construct was a generous gift of Gideon Dreyfuss(University of Pennsylvania).

Cell Cultures and Transfections

Cell cultures were maintained in standard conditions in DMEMsupplemented with 10% (v/v) bovine calf serum (Life Technologies).Depletion of U2AF subunits and U2AF35 isoforms with small interferingRNAs (siRNAs) and splice-switching oligonucleotides (SSOs), were carriedout following a time course experiment that established depletion levelsof each isoform. Oligo(ribo)nucleotides and siRNAs are listed in TableS1. Transfections were carried out in 6- or 12-well plates usingjetPRIME (Polyplus) according to manufacturer's recommendations. Thecells were harvested 48 hrs after the second hit, except for thoseexposed to IR, which received a single hit. For SF3B1 depletion, HEK293cells were exposed to a siRNAs mixture (S23850, S23852, and S223598(LifeTechnologies)) and were harvested 48 hrs later.

RNA-Seq

Analysis of differential exon usage was performed using DEXSeq (v.1.12.1), based on q-values less than 0.05. Differential gene and isoformexpression between sample sets was analyzed with Cufflinks (v. 2.1.1),which normalizes the reads using a fragments per kilobase of exon modelper million reads measure. Selection of significantly differentiallyexpressed genes was made on the basis of FDR-adjusted P-values (q<0.05).

NSE Expression in Human Tissues and Cell Lines

The FirstChoice human total RNA survey panel containing total RNAsamples from 19 different tissues was purchased from LifeTechnologies.Each tissue sample contained a pool of RNAs from different donors.Lymphoblastoid cell lines were exposed to cold and heat shock. Total RNAsamples were reverse transcribed with the Moloney murine leukemia virusreverse transcriptase (Promega) and random hexamer or oligo-d(T)primers. cDNA samples were amplified using primers shown in FIG. 20.Total RNA extracted from leukocytes from bone marrow samples of randomlyselected patients with acute myeloid leukemia or chronic myelomonocyticleukemia was reverse transcribed with random hexamer primers. The studywas approved by the National Research Ethics Service (UK) CommitteeSouth West.

Splice-Switching Oligonucleotides

SSOs were designed to maximize interactions with single-stranded regionsand avoid secondary structures predicted by Mfold. All SSOs werepurchased from Eurofins, diluted in water and their aliquots were storedat -80° C. All transfections were carried out with jetPRIME (Polyplus)according to manufacturer's recommendations.

Exposure of Cell Cultures to Ionizing Irradiation

(Mock)-depleted HEK293 cells were exposed to IR 48 hours after the firsthit using a Gulmay Medical (X-Strahl) D3225 Orthovoltage X-ray system ata dose-rate of 0.63 Gy/min at room temperature. The actual dose rate wasmonitored by a constancy meter. Cells were harvested as indicated infigure legends.

Immunoblotting

Antibodies against ATM (D2E2), ATM-pS1981 (D6H9), CHEK2 (D9C6) andCHEK2pThr68 (C13C1) were purchased from the Cell Signaling Technology,Inc. RBM39 antibodies were purchased from Thermo Fisher Scientific(PA5-31103). Antibodies detecting X-press tag, U2AF35, U2AF65, andtubulin were used. SF3B1 immunoblotting was performed with mousemonoclonal anti-SAP155 antibody (D138-3, MBL). Preparation of celllysates and immunoblotting was carried out.

TABLE 1 U2AF35-dependent transcripts are more common than expected amonggenes involved in cancer-associated gene fusions and recurrentchromosomal translocations Overlap Overlap with with Number U2AF35-P-value/rep- U2AF35- P-value/rep- of sensitive resentation sensitiveresentation Database Source Genes exons² factor³ transcripts² factor³ChimerDB [69] 1187 66 P < 0.00004/1.7 204 P < 0.02/1.1 2.0 Genes [70]300 19 P < 0.006/1.9 56 P < 0.05/1.2 involved in recurrent structuralabnormalities in cancer ¹Gene list downloaded on 2 Apr. 2014. ²Exon- andgene-level analysis of RNA-Seq data was carried out for 23,263 genesusing DEX-Seq and Cufflinks, respectively. ³Number of overlapping genesdivided by the expected number of overlapping genes drawn from twoindependent groups. A representation factor > 1 indicates a greateroverlap than expected of two independent groups, the value < 1 indicatesless overlap than expected. P-values were derived by hypergeometrictests.

Example 2 Antisense Macrowalk Targeting a Regulated Nonsense-MediatedRNA Decay Switch Exon in the ATM Gene Summary

ATM is an important cancer susceptibility gene that encodes a criticalkinase of the DNA damage response (DDR) pathway. ATM deficiency resultsin ataxia-telangiectasia (A-T), a rare genetic syndrome exhibiting ahigh susceptibility to lymphoid malignancies. ATM expression is limitedby a nonsense-mediated RNA decay (NMD) switch exon (termed NSE) locatedin intron 28, which is tightly controlled by the spliceosome. NSEinclusion in mature transcripts can be modulated by splice-switchingoligonucleotides (SSOs), but their optimal targets in the intron areunknown and their delivery to lymphoid cells has not been tested. Here asystematic search for efficient SSOs targeting intron 28 to identify NSEactivators and inhibitors was employed. Discovery of these antisensecompounds was assisted by a segmental deletion analysis of intronictransposed elements, revealing NSE repression upon removal of a distantantisense Alu and NSE activation upon elimination of a long terminalrepeat transposon MER51A. Efficient NSE repression upon SSO deliverywith chitosan-based nanoparticles to embryonic and lymphoblastoid cellswas also demonstrated, opening a possibility for NSE-mediated modulationof ATM expression in cancer and A-T. Taken together, these resultshighlight an important role of transposed elements in regulating NMDswitch exons and the power of intronic SSOs to modify gene expression.

Introduction

Eukaryotic genes contain intervening sequences or introns that need tobe removed by a large and highly dynamic RNA protein complex termed thespliceosome to ensure accurate protein synthesis. The cell requiresexcessive energy and time to complete transcription of intron containingprecursor messenger RNAs (pre-mRNAs) from at least a quarter of thehuman genome and also needs to synthesize non-coding RNAs and >200different spliceosomal proteins to achieve this task. Although onceregarded a ‘selfish’ or ‘junk’ DNA, introns are now recognized ascritical functional components of eukaryotic genes that enhance geneexpression, regulate alternative RNA processing, mRNA export and RNAsurveillance. They are also an important source of new gene-coding and-regulatory sequences and noncoding RNAs, including microRNAs andcircular RNAs. Their removal process is tightly coupled withtranscription, mRNA export and translation, with most human intronseliminated from pre-mRNA co-transcriptionally, however, their potentialas targets for nucleic acid therapy is only beginning to be unleashed.

Spliceosomes assemble ad hoc on each intron in an ordered manner,starting with recognition of the 5′ splice site (5′ss) by U1 smallnuclear RNP or the 3′ss by the U2 pathway. In addition to traditionalsplice site recognition sequences (5′ss, branch point, polypyrimidinetracts and 3′ss), accurate splicing requires auxiliary sequences orstructures that activate or repress splice sites, known as intronic orexonic splicing enhancers or silencers. These elements allow genuinesplice sites to be recognized among a vast excess of cryptic orpseudo-sites in eukaryotic genomes that have similar sequences butoutnumber authentic sites by an order of magnitude. Activation ofcryptic splice sites can introduce premature termination codons (PTCs)in translational reading frames that may lead to genetic disease. Suchtranscripts are usually recognized by a NMD pathway and downregulated.However, cryptic exons and NMD have also an important role incontrolling the expression of naturally occurring transcripts and fordifferentiation stage-specific splicing switches, as exemplified byterminal stages of hematopoiesis. In addition, cryptic splice sites maypermit unproductive or partial spliceosome assemblies that may competewith natural splice sites, facilitating their accurate selection at asingle-nucleotide resolution. Cryptic splice sites activating such‘pseudo-exons’ (also known as ‘poison’ or ‘NMD switch’ exons) that limitgene expression and regulate the pool of mRNA isoforms could thusprovide interesting targets for nucleic acid therapeutics, however,exploitation of such approaches is in its infancy.

Splice-switching oligonucleotides (SSOs) are antisense reagents thatmodulate intron splicing by binding splice-site recognition orregulatory sequences and competing with cis- and trans-acting factorsfor their targets. They have been shown to restore aberrant RNAprocessing, modify the relative abundance of existing mRNA isoforms orproduce novel splice variants that are not normally expressed by thecell. Most SSOs employed in pre-clinical and clinical development havetargeted exonic sequences. Functional intronic SSOs are more difficultto identify, unless SSOs block access to intronic cryptic splice sitesactivated by a disease-causing mutation. First, a large fraction ofintronic sequences may not affect RNA processing, despite the wealth ofintronic auxiliary splicing motifs in the human genome. In addition,their identification is costly and inefficient in long introns. Mostexonic SSOs designed to induce exon skipping have usually a desiredeffect. For example, most SSOs systematically covering SMN2 exon 7stimulated exon skipping, a prerequisite for antisense therapy of spinalmuscular atrophy, however, ˜20% increased exon inclusion. By contrast,stimulation of intron splicing was found only for ˜10% of SSOs targetingINS intron 1 while the majority failed to show this effect.Identification of effective SSOs may be facilitated by global pre-mRNAfolding and ultraviolet crosslinking and immunoprecipitation studiesthat identify binding sites for components of the spliceosome or theexon junction complex. However, these binding sites may not reflectoptimal antisense targets and their resolution may not be sufficient.Thus, a search for intronic SSOs with desired effects on RNA processingremains challenging.

The RNA-Seq studies have recently revealed activation of a NMD switchexon (termed NSE) deep in ATM intron 28 in cells depleted of eachsubunit of the auxiliary factor of U2 small nuclear RNP (U2AF). U2AFbinds to polypyrimidine tracts coupled with highly conserved 3′ss AGdinucleotides at intron ends and this binding promotes U2 recruitment tothe branch site and formation of lariat introns. However, the recentidentification of a large number of exons that were activated in cellsdepleted of each U2AF subunit (U2AF35 and U2AF65) and exhibited adistinct 3′ss organization suggested that a subset of both canonical andNMD switch exons is repressed by U2AF, similar to exon-repressing and-activating activities found for a growing number of RNA bindingproteins. The NSE levels were responsive to knockdown of additionalsplicing factors involved in 3′ss recognition and were influenced by twonatural DNA variants (rs4988000 and rs609261) located in the NSE itselfand its 3′ss, respectively. SSOs that modulate NSE inclusion levels inthe ATM mRNA by targeting NSE and its competing pseudoexon in the sameintron have also been identified. The ATM NSE provides an interestingand promising target for anticancer therapy for several reasons: (i) theATM kinase is activated in response to double-strand breakage,mobilizing an extensive signaling network with a broad range of targets,influencing cellular sensitivity to DNA-damaging agents; (ii) theU2AF-regulated exon usage in the ATM signaling pathway was centered onthe MRN/ATM-CHEK2-CDC25 axis and preferentially involved transcriptsimplicated in cancer-associated gene fusions and chromosomaltranslocations; and (iii) the ATM NSE activation limits ATM expressionin cells lacking each U2AF subunit. However, optimal NSE SSOs areunknown and their delivery to lymphoid cells has not been tested.

In the present study, SSOs covering the entire intron 28 weresystematically screened and additional SSOs that activate or repress NSEand could be exploited as putative NSE-based ATM inhibitors andactivators in therapeutic strategies were identified. Distant transposedelements in the same intron that influence NSE inclusion were alsoidentified. Finally, efficient NSE repression upon SSO delivery toembryonic and lymphoblastoid cell lines using chitosan-basednanoparticles was also shown.

Materials and Methods Plasmid Constructs

Reporter constructs containing full ATM intron 28 and flanking exonswere cloned in the HindIII/XbaI site of pCR3.1 (Invitrogen) usingamplification primers ATM26 and ATM27 (Table 2). Deletion constructs(FIG. 16) were obtained by overlap extension PCR with mutagenic primers(Table 2). Hybrid ATM minigenes were prepared by cloning ˜0.9-kbamplicons containing NSE and exon 29 into XhoI/XbaI sites of the U2AF1construct. Plasmids were propagated in E. coli (DH5α) and plasmid DNAwas extracted with the Gene JET Plasmid Miniprep kit (ThermoScientific).Full inserts were sequenced to confirm the identity of intended changesand exclude undesired mutations.

Splice-Switching Oligonucleotides (SSOs)

To test SSOs with both endogenous and exogenous pre-mRNAs, SSOs weredesigned to avoid transposed elements in intron 28. Transposons wereconfirmed in sequences of the constructs using RepeatMasker. The SSO GCcontent was at least 24% (mean 31%) and their average length was ˜20 nt.The SSOs comprehensively covered three unique regions in ATM intron 28(termed A, B and AN, FIG. 17), avoiding only homopolymeric tracts. SSOs(Eurofins) were modified at each ribose by 2′-O-methyl and by aphosphorothioate at each end linkage to ensure adequate stability forthe ex vivo screening. SSOs were diluted in double distilled water andquantified using Nanodrop (ThermoScientific). Their normalized aliquotswere stored at −80° C.

Determination of PU Values

The PU (probability of unpaired) values estimate RNA single-strandednessusing the equilibrium partition function by considering all possible RNAstructures of short sequences, permitting their comparison at eachnucleotide position. Higher PU values indicate a highersingle-strandedness of an RNA motif. The PU values were computed asdescribed using the three intronic regions and their 30-nt flanks as aninput. PU values for each position of an SSO target were averaged andcorrelated with SSO-induced NSE inclusion levels.

Preparation of Stearylated Trimethyl Chitosan

Trimethyl chitosan, originally derived from ultrapure chitosan obtainedfrom Agaricus bisporus, was provided by KitoZyme (Belgium).

Purified products had the number average molecular weight (Mn) of43.3±5.5 kDa and the polydispersity index (Mw/Mn) of 2.4±0.3, asdetermined by gel permeation chromatography in a 0.33 M NaCH₃COOH/0.28 MCH₃COOH eluent at a flow rate of 1 mL/min. The degrees of acetylationand quaternization, determined by the Fourier-transform infraredspectroscopy and 1H-nuclear magnetic resonance spectroscopy (¹H NMR),respectively, were 11.1±0.9% and 30.1±4.6%. Trimethyl chitosan wasfunctionalized with N-succinimidyl stearate (Santa CruzBioTechnologies), achieving a final degree of substitution of 2.1±0.6%(mol %), as determined by 1H NMR.

Formation of Nanocomplexes

The nanocomplexes were prepared by mixing equal volumes (30 μL) of SSOand polymer solutions. Briefly, SSOs were diluted in buffer A (20 mMHEPES, pH 7.3, 5% (w/v) glucose) and supplemented with 1 M Na₂SO₄ to afinal concentration of 50 mM. Both the polymer and SSO solutions wereheated at 60° C. for 5 min before mixing with vortex at 1,000 rpm for 15s. The tested complexes were prepared with molar ratios of quaternizedamines (N) to phosphate groups (P) of 20, 40 and 80, as previouslyoptimized, and had a hydrodynamic diameter between 110-130 nm for N/Pratios between 20-80. The complexes were allowed to stabilize for 30 minat room temperature before adding to a 240 μL of the culture medium(DMEM) without serum and antibiotics. Final concentration of SSOs inchitosan-containing cultures was 300 nM. Twenty four hours aftertransfections, 300 μL of the culture medium with serum/antibiotics wasadded. The cells were harvested 24 hrs later.

Cell cultures and transfections. HEK293 and lymphoblastoid VAVY cellswere maintained in standard culture conditions in DMEM supplemented with10% (v/v) bovine calf serum. Cells were seeded at 70% confluency 24 hrsprior to transfections. Transfections of wild-type and deletionconstructs were carried out in 12- or 24-well plates using jetPRIME(Polyplus) according to manufacturer's recommendations. The cells wereharvested 24 hrs later for total RNA extraction. Each SSO wastransfected with or without the full-length ATM construct at 50 nM andcells were harvested 48 hours later for RNA extraction.

Analysis of spliced products. RNA samples were isolated usingTRI-reagent (Ambion). Total RNA samples from chitosan experiments wereextracted with the RNeasy kit (Qiagen). RNA was quantified and 1 μg oftotal RNA was reverse transcribed with the Moloney murine leukemia virusreverse transcriptase (Promega) and random hexamer or oligo-d(T)primers. Exogenous cDNA samples were amplified using primers PL4 andATM-F and endogenous products were amplified with primers ATM-F andATM-R (Table 2). Spliced products were separated on agarose andpolyacrylamide gels and their signal intensities were measured.Statistical analysis was carried out with Stat200 (BioSoft, UK).

TABLE 2  Oligonucleotide primers SEQ ID Primer 5′-3′ sequence NO:Cloning primers ATM26 ataaagcttcttgttataaggttttgattcc 1 ATM27atatctagatgtacataccctgaaaagtcac 2 RT-PCR primers PL4 agtcgaggctgatcagcgg3 ATM-F gagggtaccagagacagtgggatggc 4 ATM-R ggctcatgtaacgtcatcaat 5Mutagenic primers del-1F atacaatttaccataatttacttttgaattatgtt 6 del-1Raagtaaattatggtaaattgtatcatacattag 7 del-2Fccttgccagaccagtttcctagttatctatattgaac 8 del-2Rtaactaggaaactggtctggcaaggtggctta 9 del-3Fcttcaagggaccttggccgggtgcggtggct 10 del-3Rgcacccggccaaggtcccttgaagtttatctaa 11 del-4Facacaaacaaagcttaggtttctacttgtcaccttcta 12 del-4Ragaaagaaacctaagctttgtttgtgtgttttatacaa 13 del-5Ftgcctcatttacgtcatacaacttaatgatagacct  14 del-5Rttaagttgtatgacgtaaatgaggcagggcaa 15 del-6Ftgatacaatttacctcatacaacttaatgatagacct 16 del-6Rattaagttgtatgaggtaaattgtatcatacattag 17 2′-O- methyl/ PTO SS_(s) ^(a) A2aacuuaaagguuauaucuc 18 A4 uauaaauacgaauaaaucga 19 A8 cauggguuggcuaugcuag20 A9 caacacgacauaaccaaa 21 A10 aagccaaucagagggagaca 22 A11aacauuucuauuuaguuaaaagc 23 A15 ucguguauuacaacaguuaa 24 A16caaccaguuugcauucgu 25 A17 uuaguauuccuugacuuua 26 A18uucuguacacuguuuaguauucc 27 A19 gaagagggagugaagguu 28 A20aaagcuuggugagauuga 29 A21 uuucuugaaaaguggaaagcuug 30 A22uggaaugagggacgguuguuuuuc 31 A23 gguaugagaacuauagga 32 A24aaacaaacagcaggguau 33 A25 gguaauaagugucacaaa 34 A26 guaucauacauuagaagg35 B1 ucaaaaguaaauuauggucu 36 B2 gacugguaaauaauaaacauaauuc 37 B3aaauguauacuggagaagacu 38 B4 auauauuagagauacaucagcc 39 B5gacaaacauuuaaugaauacucaa 40 B6 uugacuccuucuuuugacaaacau 41 B7uuuaaauccuuccuuacuu 42 B8 gauuauaaaacaaacgaagc 43 B10uguuuuaauauaaguugcuucaa 44 B11 uguggggugaccacagcuu 45 B12ucccuuacuuauauccaa 46 B13 ccaaguuugguuacuuauc 47 B14gaaguuuaucuaauauugacc 48 AN1 ggucuaucauuaaguuguauga 49 AN2uuaaauaagacuucaggucua 50 AN3 uuagagaaucauuuuaaauaagac 51 AN4cuuaauccaauucuucaauuuuag 52 ^(a)PTO, phosphorothioate

Results

SSOs targeting either 3′ or 5′ss of the NSE efficiently repress thisexon in a haplotype dependent manner. To facilitate identification ofoptimal intronic SSOs that activate NSE, splicing reporter constructswith the entire ATM intron 28 (FIG. 16A) were first prepared. Theconstruct was obtained by PCR using the HEK293 DNA as a template. Thereference sequence (hg19) of intron 28 is 3,100 nt long, which issimilar to the average human intron. Transposed elements occupy ˜64% ofintron 28, filling completely its middle part, except for a ˜350 ntregion in the 5′ half of the intron and exonic flanks (FIG. 16A).Plasmid DNA sequencing revealed the same organization of transposedelements without any additional transposon copies. It also showed the Cand G allele at rs4988000 and rs609261, respectively, indicating thatthe construct contains the haplotype most permissive for NSE inclusionin the ATM mRNA. After transfections into HEK293 cells, total RNA wasextracted and reverse transcribed prior to amplification with a vectorprimer PL4 (Table 2) and an exon primer (FIG. 16A). Examination ofspliced products showed that most transcripts entirely lacked intronicsequences (NSE-) whereas ˜36% mRNA contained NSE (FIG. 16B, lane 1), afraction slightly higher than for a hybrid reporter reported previously.

To determine the importance of transposed elements for NSE inclusion,each transposon from intron 28 was individually deleted usingoverlap-extension PCR (deletions 1-5, FIG. 16A). A large middle part ofthe intron was also deleted along with all transposons, leaving the NSEand its upstream sequences intact (˜75% of the intron, deletion 6).Transfection of validated mutated constructs, which all had identicalgenotypes to the wildtype construct at rs4988000 and rs609261, revealedthat the large deletion promoted NSE-containing transcripts (deletion 6,FIG. 16B). Deletion of the MER51 element increased NSE inclusion to alesser extent. In contrast, deletion of the antisense Alu inhibited NSEwhile deletion of long interspersed repeats (deletions 3 and 5) or aunique intronic segment (deletion 2) had no effect on NSE activation.The variability of NSE inclusion levels was much higher following atwo-hit knockdown of U2AF35, with a significant increase of NSE levelsmaintained only for deletion 6 (FIG. 16B), consistent with a majorstress component of NSE responses. A series of SSOs were then designedtargeting three intronic regions that have unique sequences in thegenome (termed A, B and AN) while avoiding a predicted branch siteupstream of NSE (FIG. 17A, Table 2). Each SSO was modified with2′-O-methyl at each ribose and phosphorothioate at each end linkage toensure their RNase H resistance and sufficient stability in transienttransfections. As positive and negative controls, SSO—NSE3 was used,which was highly efficient in blocking the NSE 3′ss, and a series ofscrambled SSOs and SSOs targeting other genes, including INS and BTKwhich were not expressed in HEK293 cells, as confirmed by RNA-Seq. EachSSO was individually transfected with or without the wild-type ATMconstruct.

Measurements of spliced products revealed that SSO—NSE3 yielded the mostefficient NSE repression (FIG. 17B). About a half of tested SSOssignificantly altered NSE inclusion levels as compared to controls, withsimilar numbers of repressor and activator SSOs. The Pearson correlationcoefficient between replicate transfections was highly significant,reaching 0.88 (P<10-8); however, the overall correlation betweenexogenous and endogenous NSE levels was only 0.35 (P<0.01).

Experiments in FIG. 16 showed that the NSE inclusion is controlled bydistant splicing regulatory sequences within and outside transposons.Experimentally determined splicing enhancer and silencer motifs in theirnatural pre-mRNA context occur preferentially in single-strandedregions, suggesting that they are more accessible to RNA bindingproteins or other ligands that control exon selection. Preferentialtargeting of SSOs to unpaired regions could thus improve a search forintronic SSOs. To test this assumption, NSE inclusion levels induced byeach SSO were correlated with their average PU values (FIG. 17C). Thesevalues estimate single-strandedness of their RNA targets using anequilibrium partition function, with higher values signaling a higherprobability of single-stranded conformation. Interestingly, SSO targetswith higher average PU values tended to induce exon skipping, suggestingthat efficient blocking of unpaired interactions as far as 2 kb from theexon can impair its activation.

The experiments described above identified a small set of intronic SSOsthat activated NSE inclusion in mature exogenous and endogenoustranscripts. Since NSE can limit ATM expression through NMD, activatorand repressor SSOs could serve as tunable gene-specific inhibitors.Transient ATM repression by NSE-activating SSOs could be advantageousfor cancer treatment by inhibiting the double-strand break signalingpathway and radiosensitization.

To test if ATM SSOs can be delivered to cells that have much lowertransfection efficiency than HEK293 cells, a stearylated trimethylatedchitosan (TMC-SA) was employed. Chitosan is a natural copolymer ofD-glucosamine and N-acetyl-D-glucosamine known for biocompatibility,biodegradability and low toxicity and immunogenicity. Whentrimethylated, chitosan acquires a permanent positive charge thatimproves its solubility at neutral pH. Stearylation was found necessaryfor formation of stable nanocomplexes with SSOs and their transfectionactivity in a HeLa/pLuc705 system, which makes use of a luciferase geneinterrupted by a mutated HBB 1 intron.

Wheher TMC-SA can facilitate delivery of SSO—NSE3 into HEK293 cells wasfirst tested. FIG. 18A shows reduction of NSE levels following exposureto SSO—NSE3-TMC nanoparticles as compared to a scrambled SSO. Thisdecline was significant for the TMC-SA/SSO—NSE3 (N/P) ratios of 20 and40. The NSE decline was also apparent when comparing NSE inclusion incells exposed to uncomplexed SSO—NSE3, consistent with their significantuptake by this highly transfectable cell line. However, the reduction ofNSE levels was less efficient for TMC-SA/SSO—NSE3 than for the sameoligo transfected with jetPrime to the same cell line at a lower finalconcentration. A significant NSE repression upon exposure toTMC-SA/SSO—NSE3 nanocomplexes was observed also for a lymphoblastoidcell line where uncomplexed SSO—NSE3 failed to reduce NSE (FIG. 18B).Collectively, these results provide the first proof-of-principle that achitosan-based delivery system of intronic SSOs can repress NMD switchexons in human cells.

Discussion

This work shows the first example of transposed elements that promoteand repress activation of a NMD switch exon (FIG. 16). Alu sequencesthemselves have a propensity to exonize through 3′ss or 5′ss activationor auxiliary splicing motifs, which contributes significantly to humanmorbidity. They can also be exonized by outlying deletions and causegenetic disease, suggesting that they can promote inclusion of distantintronic sequences in mature transcripts. This is further supported by ahigher fraction of Alus that flank alternatively spliced exons thanthose spliced constitutively. Although the exact mechanism of thesedistant effects is not understood, secondary structure of these GC-richtranscripts is likely to play a major role.

Mutation-induced exonizations have been shown for all other classes oftransposed elements, including more ancient short interspersed elementstermed mammalian interspersed repeats. In the present study, an intronictransposed element with the highest similarity to MER51A (MediumReiterated frequency repeat, family 51) repressed NSE, acting as abuffer to counteract the Alu-mediated NSE activation (FIGS. 16A and16B). The ATM MER51 is relatively GC-rich (˜44%), which may facilitateintramolecular interactions with GC-rich Alus during co-transcriptionalfolding. The element contains several inverted repeats, possibly formingstable hairpins containing exposed purine-rich loops that may controlNSE inclusion (FIG. 19). About 250,000 copies of recognizable MERsequences were estimated to exist in the human genome and many werefound in mature transcripts of protein-coding genes, contributing to thediversity of protein interactions. A mutation-induced MER exonizationevent was also shown to cause Gitelman syndrome. The 3′ part of MER51 issimilar to a long terminal repeat of retroviruses (FIG. 19), whichaccount for ˜15% of disease-causing exonizations. The origin of mostMERs was placed after the decline of mammalian interspersed repeatsbefore the spread of Alus, coinciding with expansion of mammals andsuggesting that MERs may offer insight into early mammalian radiation.However, the molecular mechanisms underlying MER-mediated exonactivation are not understood and will require further studies. Takentogether, these results suggest that the interplay of transposedelements in long introns could influence inclusion levels of many NMDswitch exons, fine-tuning gene expression.

In this work, candidate sequence-specific ATM inhibitors that act bypromoting a regulated NMD switch exon critical for ATM expression werealso identified (FIG. 17). ATM inhibitors sensitize cancer cells tocytotoxic therapy that induces double-strand breaks, including localradiotherapy, which is an integral part of treatment regimens of manycancer types. Although chemical ATM inhibitors showed a great promisefor cancer radiotherapy, their undesired pharmacokinetic profiles, hightoxicity or poor efficacy have hampered their progression into theclinic. In contrast, newly identified SSOs target unique sequences inthe human genome, their mechanism of action is well-defined and they canbe delivered to cells using natural biodegradable compounds (FIG. 18).In addition, the availability of NSE-activating and—repressing SSOsprovides an opportunity to titrate gene expression more accurately thanchemical inhibitors. The approach described herein makes use ofSSO-mediated modulation of cryptic exons that activate NMD. These exonsare usually present in natural transcripts at very low levels but theirinclusion levels can be efficiently upregulated in response to variousstimuli. Recently, a gene-specific antisense inhibition of NMD employedSSOs targeting exon junction complex deposition sites, thus permittingNMD repression without relying on skipping of a PTC-containing exon. Thetwo approaches, the former relying on intronic sequence and the latterone on exonic targets, might complement each other in the future toexpand the repertoire of antisense strategies that inhibit NMD.

The average length of SSOs employed in the screening was close to theminimum for unique targets (Table 2). The short SSOs may induce moreoff-target effects than longer SSOs, which could contribute to the lowcorrelation between inclusion levels of endogenous and exogenous NSEtranscripts. Apart from the possible suboptimal target specificity,intron 28 splicing and NSE inclusion can be influenced by adjacentintrons that were absent in exogenous transcripts. In addition, intron28 splicing may not be entirely co-transcriptional and folding andfolding kinetics of RNAs transcribed from different promoters are likelyto be distinct, contributing to the low correlation. Nevertheless, thisstudy clearly demonstrates a wealth of candidate intronic target sitesfor SSOs in the human genome, consistent with a higher informationcontent of intronic auxiliary splicing sequences as compared to lowerorganisms, which have smaller introns with a lower regulatory potentialfor alternative splicing. Although SSO—NSE3 and other SSOs can repressendogenous NSE-containing mRNAs (FIGS. 17B and 17C) and NMD transcriptswith the relative abundance as low as ˜1% can contribute to the mRNAconsumption, it remains to be tested if their reduction can lead to asustained increase of ATM protein levels in normal cells. This approachmay have a potential to alleviate phenotypic consequences of leaky A-Talleles in a mutation-independent manner, especially in homozygous A-Tpatients carrying the C allele at rs609261, which facilitates 3′ssrecognition of the NSE. Finally, chitosan-based nanoparticles have beenshown to penetrate the blood-brain barrier and accumulate in cerebellumwithout affecting histomorphology, opening a possibility to deliver NSErepressors and putative ATM activators to neural cells to amelioratecerebellar symptoms of AT.

What is claimed is:
 1. A method of modulating protein expressioncomprising: a) contacting an isolated polynucleic acid polymer to atarget cell of a subject; b) hybridizing the contacted polynucleic acidpolymer to a target motif on a pre-processed mRNA transcript, wherein ahybridization of the contacted polynucleic acid polymer to the targetmotif either promotes or represses activation of a non-sense mediatedRNA decay switch exon (NSE); c) processing a mRNA transcript of thepre-processed mRNA transcript, wherein the NSE is either present orabsent in the mRNA transcript; and d) translating the processed mRNAtranscript of step c), wherein the presence or absence of the NSEmodulates protein expression.
 2. The method of claim 1, wherein theprotein is expressed from the processed mRNA transcript.
 3. The methodof claim 1, wherein the presence of the NSE downregulates proteinexpression.
 4. The method of claim 1, wherein the absence of the NSEupregulates protein expression.
 5. The method of claim 1, wherein thepolynucleic acid polymer hybridizes to a motif within ATM intron
 28. 6.The method of claim 1, wherein the polynucleic acid polymer hybridizesto a splicing regulatory motif that competes with the NSE for aspliceosomal component.
 7. The method of claim 6, wherein the splicingregulatory motif comprises a cryptic splice site or a pseudoexon.
 8. Themethod of claim 7, wherein the splicing regulatory motif comprises apseudoexon, wherein the pseudoexon is a 24 nucleotide pseudoexon located3′ to a NSE in ATM intron 28 of the pre-mRNA transcript.
 9. The methodof claim 1, wherein the polynucleic acid polymer hybridizes to a U2AF65binding site.
 10. The method of claim 1, wherein the polynucleic acidpolymer hybridizes to a motif within a transposed element, upstream of atransposed element, or downstream of a transposed element.
 11. Themethod of claim 10, wherein the transposed element is Alu or MER51. 12.The method of claim 11, wherein the isolated polynucleic acid polymerhybridizes to a target motif within Alu.
 13. The method of claim 11,wherein the isolated polynucleic acid polymer hybridizes to a targetmotif that is either upstream or downstream of Alu.
 14. The method ofclaim 11, wherein the isolated polynucleic acid polymer hybridizes to atarget motif downstream of MER51.
 15. The method of claim 1, wherein thepolynucleic acid polymer is from 10 to 50 nucleotides in length.
 16. Themethod of claim 1, wherein the NSE comprises the sequencetctacaggttggctgcatagaagaaaaag (SEQ ID NO: 57).
 17. The method of claim1, wherein the isolated polynucleic acid polymer is a NSE-repressoragent and the NSE-repressor agent binds to the NSE, a 5′ splice site ofthe NSE in ATM intron 28, or a 3′ splice site of the NSE in ATM intron28.
 18. The method of claim 1, wherein the isolated polynucleic acidpolymer is a NSE repressor agent and the NSE repressor agent binds tothe NSE within the sequence (SEQ ID NO: 58)agTCTACAGGTTGGCTGCATAGAAGAAAAAGgtagag; (SEQ ID NO: 59)tcttagTCTACAGGTTGGCTGCATAGAAGAAAAAGgtagag;  or (SEQ ID NO: 60)tctcagTCTACAGGTTGGCTGCATAGAAGAAAAAGgtagag.


19. The method of claim 1, wherein the isolated polynucleic acid polymeris a NSE-repressor agent and the NSE-repressor agent comprises an SSOselected from the group consisting of: an SSO comprising the sequencecuucuaugcagccaaccuguagacu (SSO—NSE3) (SEQ ID NO: 53), an SSO comprisingthe sequence accuuuuucuucuaugcagccaac (SSO—NSE5) (SEQ ID NO: 54), an SSOcomprising the sequence aacauuucuauuuaguuaaaagc (SSO A11) (SEQ ID NO:23), an SSO comprising the sequence uuaguauuccuugacuuua (SSO A17) (SEQID NO: 26), an SSO comprising the sequence gacugguaaauaauaaacauaauuc(SSO B2) (SEQ ID NO: 37), an SSO comprising the sequenceauauauuagagauacaucagcc (SSO B4) (SEQ ID NO: 39); an SSO comprising thesequence uuagagaaucauuuuaaauaagac (SSO AN3) (SEQ ID NO: 51), a nucleicacid analogue thereof, and combinations thereof.
 20. The method of claim1, wherein the isolated polynucleic acid polymer is a NSE-activatoragent and the NSE-activator agent comprises an SSO selected from thegroup consisting of: a PEkr/Pedel SSO, an SSO comprising the sequenceaacuuaaagguuauaucuc (SSO A2) (SEQ ID NO: 18), an SSO comprising thesequence uauaaauacgaauaaaucga (SSO A4) (SEQ ID NO: 19), an SSOcomprising the sequence caacacgacauaaccaaa (SSO A9) (SEQ ID NO: 21), anSSO comprising the sequence gguaugagaacuauagga (SSO A23) (SEQ ID NO:32); gguaauaagugucacaaa (SSO A25) (SEQ ID NO: 34), an SSO comprising thesequence guaucauacauuagaagg (SSO A26) (SEQ ID NO: 35), an SSO comprisingthe sequence uguggggugaccacagcuu (SSO B11) (SEQ ID NO: 45), andcombinations thereof.
 21. The method of claim 1, wherein the isolatedpolynucleic acid polymer comprises a sequence with at least 70%, 75%,80%, 85%, 90%, 95%, or 99% sequence identity to a sequence selected fromthe group consisting of SEQ ID NOs: 18-52.
 22. The method of claim 1,wherein the polynucleic acid polymer is modified at a nucleoside moiety,at a phosphate moiety, at a 5′ terminus, at a 3′ terminus, or acombination thereof.
 23. The method of claim 1, wherein the polynucleicacid polymer comprises an artificial nucleotide.
 24. The method of claim23, wherein the artificial nucleotide is selected from the groupconsisting of 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE),2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl(2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), 2′-O-N-methylacetamido (2′-O-NMA), a locked nucleic acid(LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a1′,5′-anhydrohexitol nucleic acid (HNA), a morpholino, amethylphosphonate nucleotide, a thiolphosphonate nucleotide, and a2′-fluoro N3-P5′-phosphoramidite.
 25. A pharmaceutical compositioncomprising: a) a non-sense mediated RNA decay switch exon(NSE)-activator agent that interacts with a pre-processed mRNAtranscript to promote inclusion of NSE into a processed mRNA transcript,or a non-sense mediated RNA decay switch exon (NSE)-repressor agent thatinteracts with a pre-processed mRNA transcript to promote exclusion ofan NSE into a processed mRNA transcript; and b) a pharmaceuticallyacceptable excipient and/or a delivery vehicle.
 26. The method of claim25, wherein the delivery vehicle comprises a nanoparticle-based deliveryvehicle.
 27. A method of treating or preventing a disease or conditionin a subject in need thereof, the method comprising: administering tothe subject a pharmaceutical composition comprising: (i) a non-sensemediated RNA decay switch exon (NSE)-activator agent that interacts witha pre-processed mRNA transcript to promote inclusion of NSE into aprocessed mRNA transcript, or a non-sense mediated RNA decay switch exon(NSE)-repressor agent that interacts with a pre-processed mRNAtranscript to promote exclusion of an NSE into a processed mRNAtranscript; and (ii) a pharmaceutically acceptable excipient and/or adelivery vehicle; wherein the disease or condition is treated orprevented in the subject by the administration of the NSE-activatoragent or the NSE-repressor agent.
 28. The method of claim 27, whereinthe disease or condition is cancer.
 29. The method of claim 27, whereinthe disease or condition is a disease or condition associated withderegulation of ATM expression.
 30. The method of claim 27, wherein thedisease or condition is a disease or condition associated with afunctional-ATM protein deficiency.